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Full text of "Chemiluminescences and degradations of b-lactam antibiotics after oxidation by potassium superoxide"

THE CHEMILUMINESCENCES AND DEGRADATIONS OF p - LACTAM 
ANTIBIOTICS AFTER OXIDATION BY POTASSIUM SUPEROXIDE 



By 

JINGSHUN SUN 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL 

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT 

OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 

UNIVERSITY OF FLORIDA 

1998 



...J 



Copyright 1998 

by 

Jingshun Sun 



This work is dedicated to my parents, and my wife, Shuang Yang. 



ACKNOWLEDGMENTS 

I wish to offer my sincere thanks and gratitude to the chairman of my committee, 
my mentor Dr. John H. Perrin, whose friendship, support and unending patience has 
made my graduate career a very enjoyable experience. 

I thank all of the graduate students in Medicinal Chemistry department for their 
interest in my work, suggestions and friendship. Thanks go to Dr. James Winefordner, 
Dr. Ian Tebbett, and Dr. Ray Bergeron for their enthusiasm and consistent support of this 
project. Special thanks go to Dr. Kenneth Sloan and Dr. Stephen Schuhnan for their 
helpfiil advice and friendship over the last several years. 

I would like to thank my family for their love and support especially my parents; 
they deserve much of the credit for making me the person I am today. Thanks to Jan 
Kallman and Nancy Rosa who helped to make Florida my home. I would also like to 
extend my thanks to all faculty, students and stuff at Chemistry department whose 
assistance is an indispensable part of this achievement. Lastly, I would like to offer my 
thanks to Shuang Yang for her love, patience and perseverance. 



IV 



TABLE OF CONTENTS 
; ' page 

ACKNOWLEDGMENTS iv 

UST OF TABLES : viii 

LIST OF FIGURES x 

ABSTRACT xvii 

CHAPTERS 

1 INTRODUCTION 1 

Reactive Oxygen Species 1 

Singlet Oxygen 1 

Superoxide Anion 4 

Oxygen Toxicity and Diseases 6 

Rheumatoid Arthritis 6 

Pulmonary Emphysema 7 

Ischemic-Reperfusion Tissue Injury 8 

Carcinogenesis 8 

Aging 9 

Neurodegenerative Disorders 10 

Chemotherapy 11 

Removal of Free Radicals by Superoxide Dismutase and Vitamin E 11 

Increasing Formation of Free Radicals 12 

Chemiluminescence and P-Lactam Antibiotics 14 

p-Lactam Antibiotics 14 

Chemiluminescence 15 

2 OBJECTIVES 32 

3 CHEMILUMINESCENT METHODOLOGY OF STATIC MODE 36 



Materials and Apparatus 36 

Experimental Methods 37 

Preparations of Stock Solutions 38 

Preparation of Luminol Solution 38 

Preparation of Hydrogen Peroxide Solution 39 

Preparation of Saturated Potassium Superoxide Solution 39 

Preparation of Phosphate Buffer Solutions 39 

4 THE CHEMICAL PROPERTIES OF POTASSIUM SUPROXIDE 47 

Comparison Studies 47 

Solvent Selection for Potassium Superoxide 48 

5 CHEMILUMINESCENCES AND p-LACTAM ANTIBIOTICS 54 

Introduction 54 

Probe of Maximum Emission Wavelength 55 

Discussion 55 

6 MECHANISTIC STUDIES 65 

The Deuteration Experiment 65 

NMR Consideration 69 

NMR Spectra of Penicillin G 70 

NMR Spectra of Penicillin V 72 

Conclusion 74 

TLC Analysis 74 

Introduction 74 

Experimental 75 

Results and Discussions 76 

HPLC Detection 78 

Experimental 78 

Results and Discussion 80 

Conclusion 80 

HPLC/ESI-MS and HPLC/APCI-MS Analysis of the Degradation of 

Penicillin G Following the Oxidation by Potassium Superoxide 81 

Introduction 81 

Experimental 84 

Results and Discussion 85 

7 SUMMARY 120 

GLOSSARY ..:..... :.......:. 123 



VI 



APPENDICES 

A 124 

B '. 142 

C 173 

D ...! 186 

E 194 

REFERENCE 199 

BIOGRAPHICAL SKETCH 207 



vii 



LIST OF TABLES 

Table page 

Table 1-1: Some deleterious effects of systems generating the superoxide radical 28 

Table 1-2: Neurodegenerative disorders associated with free radicals 29 

Table 1-3: Family of p-lactam antibiotics 30 

Table 1-4: Analytical useful chemiluminescent emitters 31 

Table 3-1: Chemicals and reagents utilized in the static studies of chemiluminescence...41 

Table 3-2: Chemical structures and sources of p-lactam antibiotics examined 42 

Table 3-2~continued 43 

Table 3-2 — continued 44 

Table 3-3: The composition of phosphate buffer solutions 45 

Table 6-1: Effects of deuterium oxide on the intensities of chemiluminescence of 

penicillin G 92 

Table 6-2: The responds of spraying the three reagents on sample PGKOj and sample 
KO2 92 

Table 6-3: The running time of TLC developments 92 

Table 6-4: The retention times of the reference compounds and sample PGKO2 93 

Table 6-5: The operating conditions of HPLC 94 

Table 6-6: The products of recombination including dimerization after penicillin G 

reacting with potassium superoxide 95 

Table 6-7: The major products of hydrolysis of penicillin G after interacting with 

potassium superoxide 96 



viu 



Table 6-8: The major products of oxidation of penicillin G after interacting with 

potassium superoxide 97 

Table 6-9: The six p-lactam antibiotics that emit no chemiluminescence 98 

Table 6-10: The seven P-lactam antibiotics that emit chemiluminescences 99 



IX 



LIST OF FIGURES 

Figure page 

Figure 1-1: Bonding in the diatomic oxygen molecule 19 

Figure 1-2: Potential energy curves for the three low-lying electronic states of 

molecule oxygen 20 

Figure 1-3: State correlation diagrams for the reactions of the three low-lying states 
of molecule oxygen with a diene to produce endoperoxide in triplet (T) and 
singlet (S) states 21 

Figure 1-4: Possible mechanism for formation of oxygen free radical during ischemic 

reperfusion 22 

Figure 1-5: Parent penicillin 23 

Figure 1-6: Parent cephalosporins 24 

Figure 1-7: Oral beta-lactam antibiotics 25 

Figure 1-8: Nonclassical p-lactam antibiotics 26 

Figure 1-9: Reaction scheme of luminol based chemiluminescence detection 27 

Figure 3-1: Schematic diagram of setup for chemiluminescent measurement in static 

mode 46 

Figure 4-1: The chemiluminescence of luminol following the oxidation by (a) HjOj 

and(b)K02 51 

Figure 4-2: The chemiluminescence of luminol reacting with KOj in CH3OH. (a) at 

starting time, (b) two hours later, (c) three hours later 52 

Figure 4-3: The correlation between time and the chemiluminescent intensities of 
luminol after repeating the injections of 50 ^1 of 1 :1 CH3CN-CH3OH solution 
containing o.l M KO2 into 2.5 ml of 10"' M luminol every 15 minutes 53 

Figure 5-1: The comparison of chemiluminescent intensities of thirteen P-lactam 

antibiotics following the oxidation by KOj 58 



Figure 5-2: The calibration curve of penicillin G 59 

Figure 5-3: The calibration curve of dicloxacillin 60 

Figure 5-4: The photocounting chemiluminescent spectrum (intensity vs. wavelength) 
of penicillin G reacting with superoxide 61 

Figure 5-5: The photocounting chemiluminescent spectrum (intensity vs. wavelength) 
of luminol reacting with hydrogen peroxide 62 

Figure 5-6: The typical chemiluminescent spectrum (intensity vs. time) of P-lactam 

antibioticals 63 

Figure 5-7: The chemiluminescent spectrum (intensity vs. time) of ampicillin 64 

Figure 6-1: The profile of chemiluminescences of penicillin G in different solutions 

with varied deuterium oxide to water ratios 100 



Figure 6-2:'H-NMR spectrum of potassium penicillin G in DjO 101 

Figure 6-3:'^C-NMR spectrum of potassium penicillin G in DjO 102 

Figure 6-4: APT spectrum of potassium penicillin G in DjO 103 

Figure 6-5:'H-NMR spectrum of the degradation products of potassium 

penicillin G in D2O after the oxidation by solid KO2 104 

Figure 6-6: '^C-NMR spectrum of the degradation products of potassium 

penicillin G in D2O after the oxidation by solid KOj 105 

Figure 6-7: 'H-NMR spectrum of potassium penicillin V in D2O 106 

Figure 6-8: '^C-NMR spectrum of potassium penicillin V in D2O 107 

Figure 6-9: APT spectrum of potassium penicillin V in DjO 108 

Figure 6-10: 'H-NMR spectrum of the degradation products of potassium 

penicillin V in D2O after the oxidation by KO2 109 

Figure 6-11: '^C-NMR spectrum of the degradation products of potassium 

penicillin V in DjO after the oxidation by KO2 110 



XI 



Figure 6-12: The schematic diagrams of TLC assays on sample PG and sample 

PGKO2 Ill 

Figure 6-13: the schematic diagrams of preparing the samples for mass 

spectroscopy by the TLC separations 112 

Figure 6-14: The FAB-mass spectrum of the sample from spot 13 113 

Figure 6-15: The chromatograms of sample PG (a) and sample PGKO2 (b). The 

buffer system: pH5.66 phosphate buffer containing 0.1% acetonitrile 114 

Figure 6-16: Schematic of major processes occurring in electrospray 115 

Figure 6-17: The total chromatograms of sample PG and sample PGKO2 116 

Figiu-e 6-18: The zoom-in chromatogram of the new oxidation products 

generated by sample PGKO2 117 

Figure 6-19: The scheme of generation of sulfoxides after penicillin G reacts with 

potassium superoxide 118 

Figure 6-20: The proposed chemiluminescent mechanism of penicillin G after 

reacting with potassium superoxide 1 19 

Figure A-1: The positive HPLC/ESI-MS/MS ofbenzylpenilloic acid 125 

Figure A-2: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic 

acid 126 

Figure A-3: The negative HPLC/ESI-MS/MS ofbenzylpenilloic acid 127 

Figure A-4: The interpretation of the negative HPLC/ESI-MS/MS ofbenzylpenilloic 

acid 128 

Figure A-5: The positive HPLC/ESI-MS/MS of penicillin G and MS/MS of m/z 335. .129 

Figure A-6: The negative of HPLC/ESI-MS/MS of penicillin G and MS/MS of 

m/z 333 130 

Figure A-7: The interpretations of the positive and negative HPLC/ESI-MS/MS of 

penicillin G 131 



^iii*=^ 



Figure A-8: The positive HPLC/ESI-MS/MS of benzylpenillic acid 132 

Figure A-9: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilhc 

acid 133 

Figure A-10: The positive HPLC/ESI-MS/MS of benzylpenicillenic acid 134 

Figure A-1 1 : The interpretation of the positive HPLC/ESI-MS/MS of 

benzylpenicillenic acid 135 

Figure A-12: The negative HPLC/ESI-MS/MS of benzylpenicillenic acid 136 

Figure A-1 3: The interpretation of the negative HPLC/ESI-MS/MS of 

benzylpenicillenic acid 137 

Figure A-14: The positive HPLC/ESI-MS/MS of benzylpenicilloic acid 138 

Figure A-1 5: The interpretation of the positive HPLC/ESI-MS/MS of 

benzylpenicilloic acid 139 

Figure A-16: The negative HPLC/ESI-MS/MS of benzylpenicilloic acid 140 

Figure A-1 7: The interpretation of the negative HPLC/ESI-MS/MS of 

benzylpenicilloic acid 141 

Figure B-1: The positive HPLC/ESI-MS/MS of the sulfoxide of penicillamine 

dimer 143 

Figure B-2: The interpretation of the positive HPLC/ESI-MS/MS of the sulfoxide 

of penicillamine dimer 144 

Figure B-3: The negative HPLC/ESI-MS/MS of the sulfoxide of penicillamine 

dimer 145 

Figure B-4: The interpretation of the negative HPLC/ESI-MS/MS of the sulfoxide 

of penicillamine dimer 146 

Figure B-5: The positive HPLC/ESI-MS/MS of benzylpenilloic acid sulfoxide 147 

Figure B-6: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic 

acid sulfoxide 148 



xui 



Figure B-7: The negative HPLC/ESI-MS/MS of benzylpenilloic acid sulfoxide 149 

Figure B-8: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilloic 

acid sulfoxide 150 

Figure B-9: The positive HPLC/ESI-MS/MS of benzylpeniUic acid sulfoxide 151 

Figure B-10: The interpretation of the positive HPLC/ESI-MS/MS of benzylpeniUic 

acid sulfoxide 152 

Figure B-11: The negative HPLC/ESI-MS/MS of benzylpeniUic acid sulfoxide 153 

Figure B-12: The interpretation of the negative HPLC/ESI-MS/MS of benzylpeniUic 

acid sulfoxide 154 

Figure B-13: The positive HPLC/ESI-MS/MS of benzylpenicillenic acid sulfoxide 155 

Figure B-14: The interpretation of the positive HPLC/ESI-MS/MS of 

benzylpenicillenic acid sulfoxide 156 

Figure B-15: The negative HPLC/ESI-MS/MS of benzylpenicillenic acid sulfoxide 157 

Figure B-16: The interpretation of negative HPLC/ESI-MS/MS of 

benzylpenicillenic acid sulfoxide 158 

Figure B-17: The positive HPLC/ESI-MS/MS of penicillin G sulfoxide 159 

Figure B-18: The interpretation of the positive HPLC/ESI-MS/MS of penicillin G 

sulfoxide 160 

Figure B-19: The negative HPLC/ESI-MS/MS of penicillin G sulfoxide 161 

Figure B-20: The interpretation of the negative HPLC/ESI-MS/MS of penicillin G 

sulfoxide 162 

Figure B-21: The positive HPLC/ESI-MS/MS of compound 366a-penicillin G 

sulfone 163 

Figure B-22: The interpretation of the positive HPLC/ESI-MS/MS of compound 

366a-penicillin G sulfone 164 



XIV {' , 



Figure B-23: The positive HPLC/ESI-MS/MS of compound 366b-isomer of 

penicillin G sulfone 165 

Figure B-24: The interpretation of the positive HPLC/ESI-MS/MS of compound 

366b-isomer of penicillin G sulfone 166 

Figure B-25: The positive HPLC/ESI-MS/MS of compound 366c-isomer of 

penicillin G sulfone 167 

Figure B-26: The interpretation of the positive HPLC/ESI-MS/MS of compound 

366c-isomer of penicillin G sulfone 168 

Figure B-27: The negative HPLC/ESI-MS/MS of compound 366c-isomer of 

penicillin G sulfone 169 

Figure B-28: The interpretation of the negative HPLC/ESI-MS/MS of compound 

366c-isomer of penicillin G sulfone 170 

Figure B-29: The positive HPLC/ESI-MS/MS of benzylpenicilloic acid sulfoxide 171 

Figure B-30: The interpretation of the positive HPLC/ESI-MS/MS of 

benzylpenicilloic acid sulfoxide 172 

Figure C-1: The positive HPLC/ESI-MS/MS of penicillamine dimer 174 

Figiire C-2: The interpretation of the positive HPLC/ESI-MS/MS of penicillamine 

dimer 175 

Figure C-3: The positive HPLC/ESI-MS/MS of compound 440 176 

Figure C-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 440.. ..177 

Figure C-5: The positive HPLC/ESI-MS/MS of compound 632 178 

Figure C-6: The interpretation of the positive HPLC/ESI-MS/MS of compound 632.. ..179 

Figure C-7: The negative HPLC/ESI-MS/MS of compound 632 180 

Figure C-8: The interpretation of the negative HPLC/ESI-MS/MS of compound 632. ..181 

Figure C-9: The positive HPLC/ESI-MS/MS of compound 642 182 



XV 



Figure C-10: The interpretation of the positive HPLC/ESI-MS/MS of compound 

642 183 

Figure C-11: The negative HPLC/ESI-MS/MS of compound 642 184 

Figure C-12: The interpretation of the negative HPLC/ESI-MS/MS of compound 

642 185 

Figure D-1: The negative HPLC/APCI-MS of benzoic acid 187 

Figure D-2: The interpretation of the negative HPLC/APCI-MS of benzoic acid 188 

Figure D-3: The positive and negative HPLC/ESI-MS/MS of compound 336 189 

Figure D-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 336.... 190 

Figure D-5: The interpretation of the negative HPLC/ESI-MS/MS of compound 336.. .191 

Figure D-6: The positive HPLC/ESI-MS/MS of compound 511 192 

Figure D-7: The interpretation of the positive HPLC/ESI-MS/MS of compound 51 1.... 193 

Figure E-1: The negative HPLC/ESI-MS/MS of compound 269 195 

Figure E-2: The interpretation of the negative HPLC/ESI-MS/MS of compound 269.... 196 

Figure E-3: The positive HPLC/ESI-MS/MS of compound 353 197 

Figure E-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 353. ...198 



xvi 



Abstract of Dissertation Presented to the Graduate School 

of the University of Florida in Partial Fulfilhnent of the 

Requirements for the Degree of Doctor of Philosophy 

THE CHEMILUMINESCENCES AND DEGRADATIONS OF p - LACTAM 
ANTIBIOTICS AFTER OXIDATION BY POTASSIUM SUPEROXIDE 

By 

Jingshun Sun 

August 1998 



Chairman: John H. Perrin, PhD ' , 

Major Department: Medicinal Chemistry 

Penicillin G is investigated as a model to study how thirteen P - lactam antibiotics 
emit chemiluminescences with different intensities after oxidation with potassium 
superoxide. The degradation products of aqueous Penicillin G, which reacts with 
potassium superoxide, are analyzed via direct infusion by HPLC - electrospray ionization 
(ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry. A 
number of products derived from the hydrolysis, oxidation, polymerization and 
chemiluminescent reaction are identified, and reaction schemes are proposed. 

A positive correlation is observed between the chemiluminescences and the 
autoxidations initiated by the singlet oxygen molecule-OjCAg). Deuteration experiments 



xvii 



reveal that the intensity of the chemiluminescence from Penicillin G in deuterium oxide is 
approximately ten times as strong as that in neutral water. It is consistent with the longer 
lifetime of singlet oxygen molecule in deuterium oxide. The results presented here 
reinforce that singlet oxygen molecules play a crucial role in structural breakage of 
protein or DNA as well as in the specific disease entities. 






xvui 



CHAPTER 1 
INTRODUCTION 



Reactive Oxygen Species 

A free radical can be defined, as any atom or molecule possessing one or more 
unpaired electrons. It can be anionic, cationic, or neutral. In biological and other related 
fields, the major free radical species of interest have been oxygen free radicals. The term 
oxygen free radicals includes the superoxide anion radical (O2" ), the hydroperoxyl radical 
(HO2 ), the hydroxyl radical (OH ), and the peroxide radical (ROO , R = Lipid). Actually 
these free radicals belong to a group of oxygen molecules called reactive oxygen species 
because they have sfronger oxidizing ability than oxygen itself Besides the oxygen free 
radicals these reactive oxygen species consist of hydrogen peroxide (HjOj), hypochlorous 
acid (HOCl), lipid peroxide (LOOH), and singlet oxygen ('Oj). 
Singlet Oxvgen 

When the configuration of the oxygen molecule orbital is described as 
KK(2ag)^(2aJ^(3ag)^(l7:u)''(l7ig)'', the ground state oxygen itself is a biradical, as shown in 
Figure 1-1. This triplet oxygen has two unpaired elecfrons, each located in a different n* 
antibonding orbital with the same spin quantum number. In accordance with Pauli's 
principle that a pair of electrons in an atomic of molecular orbital would have antiparallel 



,.• ' 



js-t.^ 



spins, this imposes a restriction on the oxidation by the ground state oxygens because the 
new electrons removed concomitantly from nonradical molecules must be of parallel spin 
so as to fit into the vacant spaces in the n* orbitals. The transition metals found at the 
active site of oxidase and oxygenase enzymes have shown the capability of accepting or 
donating one electron at a time to increase the reactivity of ground state oxygen and to 
overcome the spin restriction. Another way of enhancing the reactivity of ground state 
oxygen is to change the spin direction of one of the two electrons in the n* orbital. Two 
higher energy singlet states of oxygen molecule were found by Childe and Mecke [1] (the 
'Eg* in 1931) and by Herzberg [2] (the 'Ag in 1934). These two forms of the oxygen 
molecule had an excess energy fraction of a chemical bond, 37.5 and 22.5 Kcal mol"', 
respectively, as shown in Figure 1-2. 

Singlet OzCSg*) is readily relaxed to 02('Ag) before it has time to react with 
anything. By definition, delta singlet oxygen is not a free radical. Like oxygen free 
radicals, however, if singlet oxygen is released in biologic systems, it is capable of 
rapidly oxidizing many molecules. In vitro chemical studies of singlet oxygen show 
singlet oxygen has the same damaging effects as does the superoxide anion toward 
various biological structures including nucleic acids, proteins, and lipids [3]. 

The two principle reactions characteristic of singlet oxygen are cycloaddition and 
the 'ene' reaction [reaction (1) and (2), respectively]: 



I 4 \/'^\/ 

y — K r c^=c R 

R R 






= + R C C^ R. C 

/ \ 

R R 






R 



The formation of endoperoxides shown in reaction (1) requires a conjugated 
double bond. The formation of hydroperoxides as shown in reaction (2) requires an allyhc 
hydrogen. When the double bonds are surrounded by electron-donating groups, the 
activation energy for both reactions can be reduced to nearly zero and the rate constant 
then approaches its maximum value of about 10^ 1 mol"' s"' [4]. The formation of 
dioxetanes in singlet oxygen reactions appears to have a much higher activation energy 
and is observed chiefly when other pathways are blocked [5,6]. This is consistent with the 
predictions of the Woodward-Hoffman selection rules for concerted cycloaddition 
reactions [7]. Such orbital symmetry considerations and more quantitative calculations 
indicate that the 'Ag* component is the only one which correlates directly with the ground- 
state products in reaction (1) [7]. These correlations are illustrated by the potential energy 
curves shown in Figure 1-3. 



Superoxide Anion 

Given a single electron the ground state oxygen molecule becomes a superoxide 
anion (Figure 1-1). It is formed in almost all aerobic cells [8,9,10]. Superoxide chemistry 
differs greatly regarding whether reactions are carried out in aqueous solution or in 
organic solvents. In nonpolar envirormients superoxide is a powerful base, nucleophile, 
and reducing agent [11,12]. Superoxide can also act as an oxidizing agent, but this ability 
is only seen with compounds that can donate protons. In aqueous solution, superoxide 
undergoes the so-called dismutation reaction to form hydrogen peroxide and oxygen. The 
overall reaction can be written as 

202- + 2H^ ^ H2O2 + O2 (3) 

Under physiological conditions, even though uncatalyzed, this reaction can be very fast, 
i.e. 10^ M"' S"' [13]. The reactivity of superoxide is greatly reduced in water, and 
dismutation reaction being favored in this media. 

The early superoxide theory of oxygen toxicity had been established on the 
accumulation of evidence showing that superoxide dismutation (SOD) enzymes, which 
remove superoxide by accelerating the dismutation reaction, are of great importance in 
allowing organisms to survive in the presence of oxygen and to tolerate the increased 
oxygen concentrations [8-10, 14, 15]. Since SOD enzymes are specific for superoxide as 
the substrate, it follows that superoxide must be a toxic species. Indeed, many damaging 



effects summarizing in Table 1-1 are associated with superoxide generating systems [16]. 
However, the damage effects listed in Table 1-1 were produced by superoxide in aqueous 
solution, moreover, superoxide has proven to be relatively unreactive toward most 
biological components [17]. Therefore, it seems unlikely that superoxide alone can create 
such damage. 

Historically, it is generally accepted that the superoxide anion radical is not a 
particularly reactive species but is potentially toxic. It can be transformed into the highly 
dangerous hydroxyl radical following the metal catalyzed Haber- Weiss reaction [8]. 



TT ^ ^ transition metal ^ ^^, ^^^ 

«^02 + O.- eatalyst ^ ^^ + ^^' + ^^ (4) 



The hydroxyl radical will react with a wide range of biological molecules in its vicinity. 

Inspection of Table 1-1 shows that the metal-dependent Haber- Weiss reaction 
does not explain all, as there are several damage examples of prevented by SOD but not 
by catalase. In many other cases, for example the organism Streptococcus Sanguis, whose 
growth appears to be independent of the availability of iron [18] as it contains no heme 
compounds and lacks catalase and peroxidase, is damaged by exposure to a superoxide- 
generating system, but this damage is not prevented by scavengers of OH. Actually, there 
is no direct evidence of hydroxyl radical in any superoxide-hydrogen peroxide reacting 
enzymatic system, and the Haber- Weiss proposal is totally based on the inhibitory effects 
of SOD enzymes and that of hydroxyl radical scavengers. On the other hand the presence 



of singlet oxygen molecule Oj ('Ag) generated in the Haber - Weiss reactions is proved 
by the characteristic 1268 - nm chemiluminescence emission spectrum of single 
molecule oxygen [19]. In view of the fact that singlet Oj ('Ag) has a sufficiently long 
lifetime to diffuse in cells and selectively damage some cell constituents, singlet Oj ('Ag) 
may be the prime oxidant. 

Ironically, it is has been known since 1977 that singlet Oj ('Ag) can be produced 
either in the non - enzymatic dismutation reaction of superoxide or in the metal catalyzed 
Haber - Weiss reaction [20]. Singlet O2 (' Ag) has never drawn as much attention as the 
hydroxyl radical does due to its low concentration and the difficulty of detection. 

.. iv ' .' '• Oxygen Toxicity and Diseases 

Abnormal production of reactive oxygen species have been associated with a 
nimiber of diseases including rheumatoid arthritis, pulmonary emphysema caused by 
cigarette smoking, ischemic - reperfusion tissue injury, carcinogenesis, aging, and 
neurodegeneration disorders such as Alzheimer's Disease, Parkinson's Disease, etc. 
Conclusive evidence suggests that all cellular components appear to be sensitive to the 
reactive oxygen species damage, lipids, proteins and nucleic acids being the most 
susceptible to this injury. 
Rheumatoid Arthritis 

Rheumatoid arthritis has many characteristics of a free-radical-produced disease. 
In rheumatoid arthritis, the synovium of the joins is swollen with an inflammatory 



infiltrate, and the joint cartilage becomes eroded. Production of synovial fluid, which 
lubricates the joint, is increased, but its viscosity is decreased because of the breakdown 
of the polymer hyaluronic acid, which acts as a lubricant. This breakdown may be caused 
by oxygen free radicals produced by neutrophils that accumulate in large numbers in the 
affected joints of patients with rheumatoid arthritis [21]. Increased levels of the products 
of lipid peroxidation reactions are found both in the synovial fluid and in the plasma of 
patients with active rheumatoid arthritis [22], this indicates the involvement of hydroxyl 
radical although the exact cause is unknown. 
Pulmonarv Emphysema 

The correlation between puhnonary emphysema and a, -protease inhibitor 
deficiency has led to the hypothesis that the lung connective tissue damage is 
characteristically associated with smoking, and this lung tissue injury results from an 
impaired ability of protease inhibitors to protect lung elastin from damage caused by 
leukocyte protease, hi human, a, -pro tease inhibitor is the major serum antipro tease, and 
following bronchoalveolar lavage of normal persons, it is responsible for more than 90% 
of such antielastase activity [23]. Electron spin resonance specfroscopy has shown that 
cigarette smoke contains a variety of oxygen- and organic- based free radicals [24] and 
that these free radicals completely prevent a, -protease inhibitor activity [25]. A recent 
interesting in vitro observation demonstrates that catalase and the antioxidants 
glutathione and ascorbic acid completely prevent the removal of elastase inhibitory 
capacity of a,-protease inhibitor by cigarette smoke. 



Ischemic-Reperfiision Tissue Injury 

Ischemic-reperfusion tissue injury can occur in several tissues in addition to the 
heart [26, 27]-for example, the small intestine, gastric mucosa, kidney, liver, and skin. 
The enzyme xanthine oxidase is widely distributed among these tissues. The intestine, 
lung, and liver have particularly high levels. The intestinal mucosa is very sensitive to 
ischemic-reperfusion injury. One hour of regional intestinal ischemia produces a 
considerable increase in capillary permeability, an effect that is reduced by superoxide 
dismutase [28]. Biochemical changes during the ischemic period are thought to be the 
basis for a burst of production of firee radicals on reintroduction of molecular oxygenation 
at reperfusion-Figure 1-4 [29]. As shown in Figure 1-4, during ischemia, adenosine 
triphosphate (ATP) is metabolized to the substrate hypoxanthine, and the enzyme 
xanthine dehydrogenase is converted to xanthine oxidase by a protease activated by 
increased free calcium. On reperfusion, hypoxanthine reacts with molecule oxygen in the 
presence of xanthine oxidase to form superoxide anion radicals. In the presence of iron 
salts, superoxide anion radicals can form hydroxyl radicals, which can bring about a 
number of damages. 
Carcinogenesis 

Carcinogenesis is thought to occur in two stages. In the initiation stage, a 
physical, chemical, or biological agent directly causes an irreversible alternation in the 
molecular structure of DNA of the cell. This alternation is followed by a promotion stage, 
in which the expression of the genes that regulate cell differentiation and growth is 



altered. Oxygen free radicals play a role mostly in the promotion phase of carcinogenesis 
[30]. Hyperbaric oxygen, superoxide anion radical, and certain organic peroxides are 
tumor promoters but may also be weak complete carcinogens [31]. In contrast, many 
antioxidants are antipromoters and anticarcinogens [32]. However, the literature is not 
extensive enough to fully describe how the molecular structure of DNA is broken down 
by oxygen free radicals or other unknown oxygen species. Further investigations are 
needed. " 

Aging .^^.^ 

, The universality of aging implies that its cause is basically the same in all species. 
A free radical hypothesis of aging has been proposed. It suggests that the free radical 
produced during normal metabolism of the cell over time damages DNA and other 
macromolecules and leads to degenerative diseases, malignant lesions, and eventual death 
of the animal [33, 34]. Oxidative DNA damage is rapidly and effectively repaired. The 
human body is continually repairing oxidized DNA. An estimated several thousand 
oxidative DNA damage sites are present in the human cell every day, most of which are 
repaired [35]. A small fraction of unrepaired lesions could cause permanent changes in 
DNA and might be a major contributor to aging and cancer. The hypothesis that oxygen 
radicals play a role in aging is supported by the observation that, in general, long-lived 
species produce fewer endogenous free radicals because of their lower metabolic rate 
[36]. Long-lived animals also have more superoxide dismutase than do their short-lived 
counterparts, and animal species with the longest life-spans have the highest levels of 



10 



superoxide dismutase [37]. A consequence of the free radical hypothesis of aging is the 
concept that free radical scavenging agents might be used to prevent aging. Several 
antioxidants, including vitamin E [38] and butylated hydroxytoluene (BHT) [39] have 
been tested in animals and have yielded equivocal results. Interestingly, a study of a self- 
selected group of high dose vitamin E users who were 65 years or older showed an 
increased mortality associated with consumption of more than 1,000 lU of vitamin E a 
day [40]. The correlation between life expectancy, life-span, and dietary antioxidant 
intake in humans remains to be demonstrated. 
Neurodegenerative Disorders . 

The central nervous system (CNS) is particularly vulnerable to free radical 
damage because of the following anatomical, physiological and biochemical reasons [41]: 

1. Relative to its size, there is an increased rate of oxidative metabolic activity. 

2. There are relatively low levels of antioxidants (e.g., glutathione) and 
protective enzyme activity (e.g., glutathione peroxidase, catalase, superoxide 
dismutase). 

3. Abundant readily oxidizable membrane polyunsaturated fatty acids are 
present. 

4. Endogeneous generation of reactive oxygen species via several specific 
neurochemical reactions are possible. 

5. The CNS contains non-replacing neuronal cells, once damaged they may be 
dysfiinctional for life. 



11 



6. The CNS neural network is readily disrupted. 

The evidence for the role of free radicals in CNS disorders is generally indirect 
due to their extremely reactivity, as well as due to the general inaccessibility of the brain. 
In vivo, direct biochemical monitoring is impossible, however, numerous experiments 
still indicate the association between free radicals and several major CNS disorders as 
shown in Table 1-2. 

Chemotherapy 

The therapies based on modulation of the formation of free radicals include: 
Removal of Free Radicals bv Superoxide Dismutase and Vitamin E 

Precedents have been made to the possible protection by superoxide dismutase 
against some kinds of free radical damage in animals. Orgotein, a bovine Cu-Zn 
superoxide dismutase, has been used as an anti-inflammatory protein drug in veterinary 
practice [60]. This agent is reported to have beneficial effects in treating rheumatoid 
arthritis, Duchenne's muscular dystrophy, and radiation-induced cystitis [61, 62, 63], but 
fiirther double-blind placebo controlled studies are needed to confirm these findings [64]. 
Human recombinant superoxide dismutase has recently been produced, and trials are 
planned to evaluate its therapeutic efficacy in myocardial infraction, kidney 
fransplantation, and bronchopulmonary dysplasia in premature infants [65]. 

Vitamin E had been shovra by studies with isolated cells and animals to protect 
against damage caused by free radicals [66]. Under normal conditions, radical scavenging 



12 



by vitamin E is just one of many mechanisms involved in minimizing jfree radical-related 
tissue damage. Many of the numerous clinical studies involving large doses of vitamin E 
have not been adequately controlled, and the therapeutic benefits of vitamin E remain 
controversial [67]. 
Increasing Formation of Free Radicals 

The main emphasis in treating human disease caused by free radicals is to 
decrease the formation of free radicals or limit their reaction at critical sites within the 
body. However, the formation of free radicals might be considered as a useftil therapeutic 
strategy when the desired therapeutic effect is to damage or kill certain cells, but 
selectivity for the desired site is a priority. The various ions plus short-lived and reactive 
free radicals formed from the interaction of ionizing radiation and water have shown 
cell-killing effect [68]. Two major free radicals, hydroxyl radical and the aquated electron 
(e'«,), result from the radiolysis of water. The hydrated elecfron reacts by neucleophilic 
addition to produce radical anions, it further reacts with molecular oxygen to produce 
organic peroxide radicals: 

R + eaq ^ R (5) 

R' + O^ ^ RO2 (6) 

A surprisingly large number of anticancer drugs in use today, almost half of those 
approved drugs in the United States, can form free radicals. This fact, considered together 
with the observation that tumor cells may be deficient in the same enzymes, i.e. 



13 



superoxide dismutase and catalase, that normally protect cells from free radical damage, 
has led to suggestions that free radicals might be involved in the antitumor activity of 
some of these drugs [69]. The best evidence of a free radical involvement in the cytotoxic 
effect of an anticancer drug is from the glycopeptide antibiotic bleomycin [70]. 
Bleomycin binds to DNA and in the presence of ferrous iron and oxygen cleaves DNA. A 
free radical species, possibly the hydroxyl radical is formed in near vicinity to the DNA 
and results in its degradation [71]. 

During the past three decades, the classic fransition metal catalyzed Haber- Weiss 
reaction has met with almost universal acceptance and forms the foundation of this very 
active research field. Although the interpretation of evidence presented on this subject has 
been difficult because of the problems in identifying the free radicals, the toxicity of 
hydroxyl radicals generated by the Haber- Weiss reaction have been blamed for almost 
every disease caused by reactive oxygen species, and the chemotherapy is exclusively 
based on modulating the formation of free radicals. That the oxygen free radicals might 
be involved in human disease is not surprising in view of their existence in many 
biological systems, which have seemingly evolved elaborate protective mechanisms 
which normally effectively prevent damage by these radicals. In certain circumstances 
singlet oxygen has been shown to create similar damages to the hydroxyl radical. Few but 
sfrong evidences already support the argument that in some diseases it is singlet oxygen, 
not the hydroxyl radical which acts as a prime oxidant. This controversy needs to be 



14 



clarified in order to better understand the oxygen toxicity and to invent new therapeutic 
strategy as well as new antioxidants. 

Chemiluminescence and p-Lactam Antibiotics 

P-Lactam Antibiotics 

The P-Lactam structure of penicillin was first proposed by Abraham and Chain in 
1943, 14 years after Fleming made his first observations on the antibacterial action of 
penicillin on a plate seeded with staphylococci. It was opposed by those committed to an 
alternative thiazolidine-oxazolone structure, however, the P-lactam structure was finally 
established beyond doubt in 1945 when an X-ray crystallographic analysis by Dorothy 
Hodgkin and Barbara Low came to a successfiil conclusion[72]. Penicillin was 
pharmacologically ahead of its time. In the early 1940s, as penicillin research was just 
getting underway in earnest, the sulfa drugs were a revolutionary new concept in 
chemotherapy. The discovery of sulfa drugs, which would selectively react with bacteria, 
was a major innovation in chemotherapy [73]. By 1948 the outstanding value in medicine 
of benzylpenicillin had been firmly demonstrated, more and more scientists were 
encouraged to develop new P-lactam antibiotics. Chemical modifications of P-lactam ring 
and the introduction of specific side chains have led to a new series of compounds with 
characteristic biological activities. Due to endless efforts, a large family of P-lactam 
antibiotics is established and still grows rapidly. Representative structures of those drugs 
are shown in Table 1-3. Many P-lactam antibiotics can be classified into four classes 



IS 



according to their clinical usefulness and pharmacokinetic properties [74]: (I) parental 
penicillins (Figure 1-5), (II) parental cephalosporins (Figure 1-6), (III) oral penicillins and 
cephalosporin (Figure 1-7), (IV) nonclassical p-lactam antibiotics (Figure 1-8). The 
analytical means for particular P-lactam antibiotics are different because of their diverse 
chemical, physical, and biological properties. In the case of the analysis of penicillin G, 
many methods are adopted [75] including titration, colorimetry and UV 
spectrophotometry, florescence, HPLC, thin layer chromatography, gas-liquid 
chromatography, electrophoresis, polarography, enzyme electrodes, isotopic assay, 
biological-based microbiological assay, immunoassay, and enzyme-aided assay 
(hydrolysis). In 1991, Schulman and Perrin revealed that cephalothin has the ability to 
prolong and intensify the chemiluminescence derived from the cobalt (Il)-luminol- 
hydrogen peroxide system. It can be used as the basis for the determination of 
cephalothin in the range of 0.4-400 ^ig ml"'[76]. Post column chemiluminescent detection 
after liquid chromatography has also been used to prevent mixing problems and so 
increase the reproductivity and sensitivity. 

Chemiluminescence 

Chemiluminescence can be defined as the emission of light as a result of the 
generation of electronically excited states formed in a chemical reaction. The range of 
wavelengths of light emitted is suprisingly large-from the near ultravilet to the infra-red. 
Chemiluminescence is "cold light." The energy is released in the form of light rather than 



16 



heat. It has wider variety of color than the bioluminescence of the firefly but is far less 
efficient. Analysis by chemiluminescence has simple instrumentation, has excellent 
sensitivity, has very low limits of detection, and has great selectivity and wide dynamic 
ranges. The efficiency of a luminescent reaction is defined as the number of photons 
emitted per reacting molecule. The quantum yield is usually written as: 

cDcl = ^rxOesX<1^f (7) 

where Or is the yield of product, O^s the number of molecules entering the 
excited state and Op is the fluorescence quantum yield. High yields of excitation 
without strong visible emission are possible if Op is very low, as is the case for simple 
dioxetanes. Phosphorescence is difficult to observe under the conditions of most of the 
reactions, but in principle Op^,,, can replace Op . Sensitized chemiluminescence results 
when transfer of energy takes place between chemiluluminescent reactants and 
fluorescent acceptors. The efficiency of this energy transfer (ET) must be taken into 
account: 

^cl = <I5rX^esX<I^etX^p (8) 

where Op, is the fluorescence efficiency of the acceptor, and O^x is the efficiency of 
energy transfer. Addition of fluorescent acceptors can enhance light emission in the case 
of dioxetanes. The analytically useful chemiluminescent emitters are listed in Table 1-4. 

If a target analyte can be determined via HPLC chemiluminescence then it 
probably has one of three characteristics: 



17 



1 . it either emits chemiluminescence when mixed with a specific reagent; 

2. it catalyzes chemiluminescence between other reagents; 

3. it is suppresses chemiluminescence between other reagents. 

Since not many pharmaceuticals can give out chemiluminescence, most analysis of 
HPLC chemiluminescence are based on 2) and 3) reactions, in which luminol (5-amino-2, 
3-dihydro-l,4-phthalazinedione), reacts with oxidants like hydrogen peroxide in the 
presence of a base and a metal catalyst to produce an excited state product (3- 
aminophthalate, 3-APA) which gives off light at approximately 425 nm. When a 
compound eluting from the LC column enhances or suppresses this background light, the 
amount of light increased or decreased will represent the amount of analyte. The reaction 
system is described in Figure 1-9. Recently Garcia Campana, Baeyens, and Zhao 
reviewed chemiluminescence detection in capillary electrophoresis [92]. The combination 
of capillary electrophoresis, which is versatile and robust, and chemiluminescence-based 
reactions, which are extremely sensitive, is promising for numerous applications in fields 
such as environmental analysis and medicine. However, it is still not used as a routine 
technique. Several problems need to be solved, for instance, chemiluminescence lacks 
selectivity, the emissions of chemiluminescences depend on environmental factors; and 
the optimization of the volume and geometry of the detection cell and connecting 
hardware. 



18 



In our experiments the investigation of the behavior of the reactive oxygen 
species has always been the priority. Eleven p-lactam antibiotics have been reacted with 
potassium superoxide and the oxidation as well as hydrolysis of penicillin G have been 
examined. The deuteration experiment using penicillin G reinforces the concept that 
singlet oxygen is generated by superoxide dismutation and that singlet oxygen play an 
important role in tissue injury and related diseases. 



' - * i. 



19 



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20 




Internuclear Distance |aI 



Figure 1-2: Potential energy curves for the three low-lying electronic states of molecular 
oxygen. 



21 



X . 


A / 


-—^ S3 


/-^ s, 


^\ T, 


\. 


V So 


% . 







Figure 1-3: State correlation diagrams for the reactions of the three low-lying states of 
molecule oxygen with a diene to produce endoperoxide in triplet (T) and singlet (S) 
states. 



22 



Krhpmlfl Adenosine 

iscnemia •, Xanthine 

Inosine dehydrogenase 
Hypoxanthine 



Reperfusion 



Protease 




Xanthine 



H202< 
I Iron salts 

HO' + HO -+ O2 



Figure 1-4: Possible mechanism for formation of oxygen free radical during ischemic 
reperfusion. 



23 



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26 



Nonclassical beta-lactam antibiotics 



Nocardicin A 



Clavulanic acid 

CP-45899 

CP-47904 



Thienamycin 

Epithienamycin A 

N-acetylthienamycin 

MK-0787 

Carpet imycin 

MM-4550 

PS-4-7 



Figure 1-8: Nonclassical P-lactam antibiotics. 



In Figure 5, 6, 7, 8 the compounds with similar structures are drawn in the same block. 



27 






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30 



Table 1-3: Family of P-lactam antibiotics. 



Parental 
Penicillin 



Parental 
Cepholosporin 



Nonclassical 



Structure 



R 



HO 




K 




CHj 
CH, 



Penicillin G, R= <f ^CHj- 




Cephalothin, R,= ^ /^-CHj- 



R2=-OCOCH3 



pH 



HO — N, 







HjN 



Nocardicin A 



■>^ >*:;^"'~ 



ho' 
Clavulanic Acid Sulfones 



Thienamycin 



31 



Table 1-4: Analytical useful chemiluminescent emitters. 



Chemiluminescent 
Emitters 


^max (wavelength Region) 


Reference 


3-Aminophthalate (from 
Luminol) 


425 nm 


77 


CHaSe 


750-825 nm 


78 


CN 


383-388 nm 


79 


HCF 


475-750 nm 


80 


HCHO 


350-500 nm 


81 


HF 


670-700 nm 


80 


HSO 


360-380 nm 


82 


IF 


450-800 nm 


83 


N-methylacridone (from 
lucigene) 


420-500 nm 


84 


Na 


589 nm 


85 


NO2 


1200 nm 


86 


OH 


306 nm 


82 


Oxyluciferin (from 
luciferin) 


562 nm 


87 


Ru(bpy)3 


600 nm 


88 


S2 


275-425 nm 


89 


SF2 


550-875 nm 


90 


SO2 


260-480 nm 


91 



CHAPTER 2 
OBJECTIVES 



Investigation of the chemiluminescence following the oxidation of p-lactam 
antibiotics by potassium superoxide will facilitate a better understanding of the 
involvement of 'Oj in numerous human diseases caused by oxygen toxicity. 

Recently more and more people began to realize that the generation of OH by 
transition metal-catalyzed Haber- Weiss reaction is not enough to account for all the 
damaging effects in the biological components. It is believed that the Haber- Weiss 
reaction does not occur in biological tissues under normal conditions, but its occurrence 
during pathological states, e.g. ischaemia, is possible. Even so the Haber- Weiss reaction 
faces the competition from the Fenton type reaction to produce OH. In the meantime, the 
existence of 'Ozis indicated in the superoxide-generation systems. Mao [93] et al. utilized 
2, 2, 6, 6-tetramethyl-4-piperdone as a spin trap of electron spin resonance (ESR) 
spectroscopy to study the generation of 'Oj They observed the 'Oj spin adduct signal in 
the incubation of xanthine, xanthine oxidase and HjOj, and the depletion of each of the 
incubation components led to a sharp decrease in 'Oj generation. 'Oj scavengers like 
sodium azide inhibited 'Oj generation while the OH scavenger, ethanol, only slightly 
decrease the signal intensity. Catalase inhibited 'O2 generation and H^Oj enhanced it. 



32 



33 



They believe superoxide is capable of generating 'O2 upon reaction with HjOj, i.e. the 
Haber-Weiss reaction. Interestingly they also found that the decomposition of KO2 
generated 'O2, which is consistent with the experimental results obtained by Khan et al. in 
1970 [94], 1976 [95], 1981 [96], and 1987 [97]. However, Nilsson and Keams [98] in 
1974 challenged this conclusion with a deuteration study. They adopted the same reacting 
system as did Khan and injected DjO rather than HjO into dry dimethyl sulfoxide 
(DMSO) containing KOj, expecting increased intensity of chemiluminescence from the 
relaxation of OzC'Ag) to ^Oz because of the large differences in lifetimes in the two 
solvents: 2 jxsec in H2O vs. 20 |xsec in DjO. However, no enhancement of 
chemiluminescent emission was observed, and no oxidation of the "Oj-acceptor 
tetramethylene (TME) could be detected in this system. Their data suggested that the 
superoxide anion radical can not be a direct precursor of 'O2. Since no later evidence has 
been presented, and the deuteration experiment is a very reliable approach to prove the 
existence of 'O2, the Nilsson and Keams' study still casts doubt over the possibility of the 
generation of 'O2 from superoxide. 

Indeed, despite a tremendous amount of work, this remains one of the most 
controversial, confusing, and frustrating areas of research, and the indirect detection of 
OH or other reactive oxygen species based on the scavenger's effects or on the spin 
trap's signals is one of the major reasons. This is because OH and 'O2 are very reactive 
species, and thus are able to react with many compounds. Therefore, certain compounds 
can be spin traps for both of them, and some scavengers of OH may also be effective for 



34 



'Oj. For example, histidine can eliminate 'Oj in addition to the molecular OH radical 
[99]. 

In the current study based on some similar molecular structures, P-lactam 
antibiotics are used as models to study how the superoxide-generating system damages 
DNA molecular and protein structures. The following approaches are made to improve 
the understanding of oxygen toxicity. 

1). Evaluate KOj's capability as an oxidizer for chemiluminescence. The same 
amount of KOj and H2O2 will be used to react with luminol respectively, and under 
identical experimental conditions to check which gives the stronger chemiluminescence. 

2). Study the chemical properties of KO2 in the liquid phase. A solvent must be 
found to solubilize KOj and allow it to keep its activity as long as possible, because the 
biological nature of superoxide anion has to be mimicked and handling solid KO2 is very 
dangerous. - /< 

3). Investigate whether KO2 can oxidize p-lactam antibiotics with the resultant 
emissions of chemiluminescence. This will answer the question that how and why oxygen 
toxicity can damage cell components. 

4). Design and carry out new DjO experiments to clarify the historical 
controversy. 

5). Study the mechanism of chemiluminescence of P-lactam antibiotics. A 
prediction of chemiluminescences from other compounds is desired. 



35 



6). Identify the degradation products and pathways to further confirm the 
chemiluminescent mechanism and oxygen toxicity. 

7). Try to answer the question: which plays the major role in structural breakage 
of protein and DNA, hydroxy 1 radical or singlet oxygen? 



CHAPTER 3 
CHEMILUMINESCENT METHODOLOGY OF STATIC MODE 



Materials and Apparatus 

The chemiluminescent studies were conducted with a FL-750 Spectrofluorescence 
Detector (Mcpherson, Acton, MA) in the static mode. The data was collected either by a 
Servogor 120 flatbed recorder (Norma Goerz Instruments, Elk Grove Village, IL) or by 
an IBM personal computer XT with Spectra Calc (Galactic Industries Corporation, 
Salem, NH) as the software and DT 281 1 Analog plus Digital Input / Output board (Data 
Translation Inc., Malbora, MA) as the interface. The sample was weighed accurately to 
0.1 mg by a XE-IOOA Electronic Balance (Denver Instrument Company, Arvada, 
Colorado). A Fisher Model 10 Accumet pH meter (Fisher Scientific, Pittsburgh, PA) 
measured the pH values with the sensitivity of 0.01 pH unit (or 1 mv). A Branson 
ultrasonic cleaner (Branson, Shelton, Conn.) and a Coming PC-351 Hot Plate Stirrer 
(Coming Inc., Coming, NY) was used to facilitate the dissolution of solutes with low 
solubilities. The saturated solution of potassium superoxide in 1 8-crown-6-ether- 
acetonitrile was centriftiged with a Beckman CPR Centrifuge (Beckman Instruments Inc., 
Fullerton, CA). The sources and stmctures of chemicals utilized in these experiments are 
listed in Table 3-1. 



36 



37 



The cell compartment used for the chemiluminescent detection was modified in 
order to prevent light leakage from the outside of the cell. The needle of a syringe was 
connected to a curved metal tube, which was immersed in the center of the cuvette. A 
black rubber stopper with a small hole allowing penetration of the metal tube covered the 
cuvette holder. Since it can not travel through the curved metal tube, the light does not 
enter the cuvette on withdrawal of the injection needle. Also a foam box coated with dull 
black paint inside and outside served as a "dark room" to cover the whole cell 
department. The setup is described in Figure 3-1. This light proof system was tested by 
injecting pure water into the cuvette filled with a 10* M solution of alkaline luminol. 
Even when the detector was set at its most sensitive status with the High Voltage (HV) of 
Photomutiplier (PMT) at 1000 volts (Gain = 10.0), Sensitivity at 0.003, and Time 
Constant at 0.25, no signal was observed. After injecting 3% H^Oj into the same solution, 
the photons collected by PMT was off scale. This proves that in the current system only 
the light emission inside the cuvette is detected. 

Experimental Methods 

After the detector and the recorder have been warmed up for several minutes, 2.5 
ml of the solution to be detected, was pipetted into the cuvette, and the cuvette was then 
placed into the cell holder. When the cell holder was capped by the rubber stopper and the 
"dark box" was sealed, the system was ready for injection. At least three injection were 
made to get an average of chemiluminescent peak heights or integrated areas with 



38 



reasonable reproducibility; usually the Relative Standard Deviation (RSD) was less than 
10 %. The cuvette was first washed three times with tap water, then three times with 
deionized water, finally rinsed with acetone, and dried with pressured air. The quartz 
fluorescent cuvette was fi-equently used in these experiments. The UV quartz cuvette can 
also be used, but the transparent walls must face the PMT window, and no emission 
filters must be installed so that the PMT collects the total photon emission fi-om the 
chemiluminescent reactions. Prior to every new experiment the detecting system was 
validated by a luminol-based chemiluminescent system. A 20 |xl, 3% of H2O2 was 
injected into a 2.5 ml, 10"'M of alkaline luminol solution, and a strong, broad peak was 
detected. The average peak height was 238.5 mv. The experiment parameters were: Gain 
= 7.10 (710 volts). Sensitivity = 0.1, Time Constant = 0.25, recorder Detecting Scale = 
500 mv, recorder Paper Rate = 1 cm/min. The high concentration of reactants and stable 
signal made this validation very reliable. 

Preparations of Stock Solutions 

All chemicals and reagents were used as provided without fiirther purification. 
Preparation of Luminol Solution 

The 0.001 M luminol solution was prepared by the following procedures: 88.58 
mg of luminol was weighed and then dissolved in a mixture of about 150 ml deionized 
water and 6.25 ml of 5 N sodium hydroxide solution with stirring. This solution was 
transferred into a 250 ml volumetric flask and the volume was adjusted to 250 ml. After 



39 



shaking the flask gently to achieve a homogeneous mixture, the stock solution was stored 
in refrigerator. The high pH of this solution benefited the further dilutions as no 
additional base was needed to keep luminol in alkaline media. 
Preparation of Hydrogen Peroxide Solution 

The 3 % H2O2 solution was prepared by pipetting 10 ml of 30 % H2O2 solution 
into a 100 ml volumetric flask then adjusted to volume with deionized water. 
Preparation of Saturated Potassium Superoxide Solution 

The KO2 was weighed quickly and put into dry acetonitrile, then 18-crown-6-ether 
was added in excess to solubilize the KOj powder. After gentle stirring, the solution was 
stored overnight in the refrigerator. The acetonitrile solution, containing excess KO2 and 
crown ether, was centrifuged at 2500 rpm for 10 minutes at room temperature to leave a 
saturated solution. Acetonitrile was chosen as the solvent because its polarity enhanced 
the solubility of ionic species, and also because it is miscible with the aqueous solutions. 
Preparation of Phosphate Buffer Solutions 

Based on the Henderson-Hasselbalch equation. Table 3-2 was developed to 
prepare phosphate buffer solutions. To make a buffer with pH range from 3.7 to 9.2, 
certain amounts of potassium phosphate monobasic and sodium phosphate dibasic were 
weighed corresponding to the clost pH value in Table 3-2, and dissolved in about 450 ml 
deionized water. While the solution was stirred by a hot plate stirrer until the dissolution 
was completed, the pH change was monitored by a pH meter and small amounts of dilute 



40 



solutions of sodium hydroxide or hydrochloric acid were added to adjust the pH value to 
the desired one. Then, the solution was transferred into a 500 ml volumetric flask and 
made to volume. After gently shaking the solution, the pH value of a small portion of it 
was measured to confirm the final pH. 

All the P-lactam antibiotic solutions were made just before the detection 
experiments to minimize the effects of hydrolysis, and all the stock solutions were shaken 
before being utilized. 



41 



Table 3-1 : Chemicals and reagents utilized in static studies of chemiluminescence. 



Millipore": Millipore Corp., Bedford, MA. 

Aldrich^ Aldrich Chemical Company, Inc., Milwaukee, WI 53233. 

Aldrich": Aldrich Chemical Company, Milwaukee, WI53201. 

Fisher'': Fisher Scientific, Fair Lawn, NJ 

Mallinckrodf : Mallinckrodt, Inc., Paris, Kenturkey. 



Class 


Chemical or Reagent 


Source 


Grade 




Water 


Milli-Q , Ultra-pure water 
system. Millipore^ 


Deionized 




Deuterium Oxide 


Aldrich'' 


99.9 atom % D 




Methanol 


Fisher 


HPLC grade 


Solvent 


Reagent Alcohol 


Fisher^ 


HPLC grade 




n-Butanol 


Fisher 


Certified A. C. S. 




Acetone 


Fisher^ 


Pesticide grade 




Methylene Chloride 


Fisher'' 


Certified A. C. S. 




18-Crown-6-Ether 


Aldrich' 


99% 


Acid and 
Base 


85 % Phosphoric Acid 


Fisher 


Certified A. C. S. 


Hydrochloric Acid (2N) 


Fisher'' 


Certified 




Sodium Hydroxide (5N) 


Fisher" 


Certified 




Potassium Phosphate Monobasic 


Fisher'' 


Primary standard 




Sodium Phosphate Dibasic 


Fisher'' 


Certified A. C. S. 




Sodium Phosphate Tribasic 


Mallinckrodt' 


Analytical Reagent 


Buffer 


Buffer Solution Concentrate pH4.00 


Fisher'' 


Certified 




Buffer Solution Concentrate pH6.00 


Fisher" 


Certified 




Buffer Solution Concentrate pH7.00 


Fisher" 


Certified 




Buffer Solution pH 10.00 


Fisher" 


Certified 


Oxidant 


30 % Hydrogen Peroxide 


Fisher" 


Certified A. C. S. 




Potassium Suproxide 


Aldrich" 


N/A (Powder) 



42 



Table 3-2: Chemical structures and sources of P-lactam antibiotics examined. 



Beta-Lactam 
Antibiotic 



Stucture 



Source 



Azetidinone 



Sulbactam 



Clavulanic Acid 



Cephalothin 



HN — I 

P 



j/ 







V 



OH 





Cefotaxime 




Aldrich" 



Fluka' 



Beecham'' 



LiUy 



Sigma' 



Table 3-2~continued 



43 



Beta-Lactam 
Antibiotic 


Structure 


Source 


Penicillin G 


OH 


Fluka" 


Penicillin V 


"^^ 


Sigma'' 


Ampicillin 


o_/NH 
OH 


Interchem*^ 


Amoxicillin 


OH 


Sigma' 


Piperacillin 


HN-^X^.H 


Sigma' 



44 



;-pA 



Table 3-2 — continued 



Beta-Lactam 
Antibiotic 


Structure 


Source 


Dicloxacillin 




Sigma' 


Methicillin 




Sigma' 


Hetacillin 


OH 


Beecham' 



Aidrich": Aldrich Chemical Co., Inc. Milwaukee, WI. 

Fluka*": Fluka Chemical Corp. Milwaukee, Wl 

Beecham'; Smithkline Beecham Pharmaceuticals, Philadelphia, PA. 

Lilly*: Eli Lilly and Company, Indiapolis, Ind. 

Sigma': Sigma Chemical Co., St. Louis, MO. 

Interchem': Interchem Corporation, Paramus, NJ. 



45 



Table 3-3: The composition of phosphate buffer solutions. 



85 % H3PO4, ml 


KH2PO4, g 


Na^HPO^, g 


Na3P04, g 


Measured pH 


0.99 


6.794 






2.5 


0.61 


6.7983 






2.75 


0.34 


6.8023 






3.05 


0.12 


6.8095 






3.43 


0.05 


6.8103 
6.8133 






3.7 
4.43 




6.7312 


0.0453 




4.73 




6.463 


0.107 




5 




6.0688 


0.25 




5.42 




5.232 


0.5488 




5.84 




4.6136 


1.0166 




6.17 




2.7122 


1.415 




6.56 




1.1775 


1.9653 




7.06 




0.5317 


2.1866 




7.48 




0.1553 


2.3198 




8.04 




0.0256 


2.3677 
2.363 




8.77 

9.21 




! -. 


2.3614 


0.0771 


9.59 






2.2292 


1.868 


10.2 






2.0447 


0.4343 
3.1617 


10.64 
11.79 



*The final volume of all buffer solution is 500 ml, and ionic strength is 0.1. 






Black rubber stopper 




46 



Curved matel tube, 
connected to syringe 



CeU holder 
- Cuvette 



PMT 



Light proof box 



Figure 3-1: Schematic diagram of setup for chemi luminescent measurement in static 
mode. 



CHAPTER 4 

THE CHEMICAL PROPERTIES OF 

POTASSIUM SUPROXIDE 

Comparison Studies 

Since it is a well-documented oxidant for the chemiluminescent reactions of 
luminol, H2O2 was used as a reference to evaluate K02's capability as an oxidizer 
producing chemiluminescence. 

At the same concentration (1 M), 40 \x\ each of aqueous H2O2 solution (a) and 
K02(b) were reacted with 2.5 ml of lO'^M luminol. The chemiluminescences were 
recorded by a flatbed recorder. The signal profiles are shown in Figure 4-1. At least three 
injections were used to obtain the average peak heights for (a) and (b). The intensities in 
term of peak heights can be calculated as 

I. = 53.5 / 100 X 0.003 X 100 mv = 0.1605 mv 

lb = 70.5 / 100 X 0.1 X 500 mv = 35.25 mv 

lb /I, = 35.25 7 0.1605=219.6 
Here, 53.5 is the peak height in centimeters, 100 is the scale height in centimeters which 
corresponds to the signal scale of 100 mv, and 0.003 is the sensitivity of the 
photomultiplier tube. Figure 4-1 clearly demonstrates that the chemiluminescence from 



47 



48 



the reaction of luminol and KO2 emitted faster, disappeared faster with greater increased 
intensity over the reaction with HjOj. Using the same experimental parameters, further 
examinations shown that 5.0 x 10"' M luminol was detectable with H2O2, but 5.0 x 10" 
M with KO2. The quicker chemi luminescence from the superoxide system seems more 
desirable for quantition of reactions emitting chemiluminescences, and KO2 probably has 
the greater potential to make P-lactam antibiotics chemiluminescent compounds. 

These results indicate that the reaction mechanism with KOj is different from that 
with H2O2. To increase the chemiluminescence by more than two magnitude, a significant 
lowering of activity energy is necessary. It seems that the superoxide anion decomposes 
into other reactive species. 

Solvent Selection for Potassium Superoxide 

KO2 solutions with a concentration of 0.1 M were made by dissolving 177.5 mg 
of KO2 in 25 ml of various solvents just before the chemiluminescent experiments. These 
solutions were evaluated using 10"^ M luminol injections made every 15 minutes. The 
experimental parameters were the same as in the comparison experiments, and the 
stability of chemiluminescent emissions was evaluated. 

Clearly, water is not a suitable solvent for KO2 because of its extremely high 
reactivity towards KO2. In CH3CN, KOjWas able to oxidize luminol and emit 
chemiluminescences with similar intensities and peak profiles to KO2 in H2O. However, 
the signal reproducibility was not good. Although CH3CN is miscible with HjO, the 



49 



mixing status is more variable following injection than is the mixing of the two solutions 
in water. 

CH3OH was also investigated as a solvent for KO2. The intensity of 
chemiluminescence grew abruptly, but disappeared gradually. This is probably due to the 
relative low rate of releasing KOj from CH3OH into the water. Even though the short- 
term signals were reproducible, the chemiluminescent emissions of repeated injections 
decreased significantly over time, as shown in Figure 4-2. 

As nonpolar solvents, n-butanol and methylene chloride can barely dissolve KO2, 
and no chemiluminescent signal was observed. Furthermore, these solvents are not 
completely miscible with water. 

Unfortunately, all of these solvents can not preserve the activity of KO2 over two 
hours. Even when KO2 was dissolved in the mixed solvents like CH3CN-CH3OH, 
CH3CN-H2O, and CH3OH-H2O, a similar loss of activity of KOj was observed. The 
typical correlation between the time and chemiluminescent intensity is shown in Figure 
4-3. 

To increase the solubility of KO2 in CH3CN, 1 8-crown-6-ether was added to give 
a saturated solution. After standing over night and centrifuged as described in chapter 3, 
the saturated KO2 reagent was tested by reacting with luminol. The results were very 
positive, the chemiluminescent intensity was greatly increased due to the high 
concentration of KOj, and the signal was stable for at least 24 hours. More encouragingly, 
fresh KO2 and crown ether can be added periodically to keep the KO2 amount constant. 



50 



and the reactivity of this reagent was sustained. The concentration of KO2 was found to 
be 0.4386 M as determined by atomic emission spectroscopy (Zeeman 5100, Perkin- 
Elmer, Norwalk, CT, courtesy to Dr. Bergeron). 






51 



n. 



OTJ 



xsry 









CTT 



SL 



oP 



fczQi 



Figure 4-1 : The chemiluminescence of luminol following the oxidation by (a) H2O2 and 
(b) KO2. Experimental parameters: High Voltage (HV) of Photomultiplier Tube (PMT) is 
910 volts, Time Constant is 0.25, Suppression Background is set at high, recorder speed 
is 1 cm/min. Sensitivity of PMT and Signal Scale of recorder is adjusted according to 
varied intensities. In (a) Sensitivity = 0.003, Signal Scale = 100 mv. In (b) Sensitivity = 
0.1, Signal Scale = 500 mv. 



52 



>. ' ■'.. 




Figure 4-2: The chemiluminescence of luminol reacting with KOj in CH3OH. (a) at 
starting time, (b) two hours later, (c) three hours later. 



53 







M 

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CHAPTERS 
CHEMILUMINESCENCES AND p-LACTAM ANTIBIOTIC 

Introduction 

In the last few years, quantitative studies of P-lactam antibiotics by 
chemiluminescences were based on either their enhancement [100, 101] or inhibition 
[102] of the luminol system. In this chapter the chemiluminescent emission following the 
interaction of KOj with 13 p-lactam antibiotics was investigated as a potential analytical 
tool. 

The chemiluminescent intensities of antibiotics were determined at optimized 
parameters (pH 4.5-7.0. 950 volts across the PMT). 50 ml of saturated KO^ solution was 
injected into 2 ml of 1.0 mg/ml antibiotics in 100 ml deionized water. All aqueous 
antibiotic solutions were prepared just before the detection to minimize the hydrolysis 
effects. The materials and methods were as described in the chapter 3, 

Six of the antibiotics studied gave no measurable signal, while the other seven 
emitted differently intensified chemiluminescence. The chemiluminescent intensities of 
the antibiotics are compared in Figure 5-1. The strongest chemiluminescence was 
observed with penicillin G. The linear dynamic range of 0.01-0.1 mg/ml is shown in 
Figure 5-2. Dicloxacillin in the concentration range 0.2 to 1.0 mg/ml also gave a linear 



54 



55 



relationship, as shown in Figure 5-3. 30 % H^Oj had been used to oxidize all the 
antibiotics but no signal was observed. 

Probe of Maximum Emission Wavelength 
The maximum emission wavelength of the chemiluminescence following the 
oxidation of 10 mg/ml penicillin G by the saturated KO2 reagent was measured at 535 nm 
by using a SPEX Photocounting Fluorescence Detector Courtesy of Dr. Winefordner. 
After turning off the excitation source, the fluorescence detector was ready for 
chemiluminescence determination. The injection of KO2 was made at emission 
wavelengths of 400, 450, 520, 530, 540, 600, 700, and 800 nm. The intensity observed at 
535 nm was the highest. The chemiluminescenct spectrum between 530-590 nm is shown 
in Figure 5-4. To validate the detecting system, the spectrum of 10"* M luminol (Figure 5- 
5) was observed by reacting it with 30% H^Oi, this data is consistent with the literature 
[77]. The experiments here demonstrate that the maximum emission from the reaction 
between penicillin G and KO2 is at 535 nm. 

Discussion 

There are some unique characteristics produced by the reactions of p-lactam 
antibiotics with KO2. First, in these experiments seven of the thirteen p-lactam antibiotics 
generated chemiluminescences at pH 4.5-7.0 rather in the more usual alkaline media. It 
should be remembered that these antibiotics will hydrolyze at extremely acidic or basic 
pH values [103]. Second, the maximum emission wavelength of chemiluminescence 



56 



following the oxidation of penicillin G by KO2 was observed at 535 nm, which is 
different from the 425 nm of 3-aminophthalate (from luminol) [77], 275-425 nm range of 
S2 [89], 260-480 nm of SO^ [91]; but is closer to the 420-500 nm range of N- 
methylacridone (from lucigene) [84] or 562 nm of oxyluciferin (from luciferin) [87]. 
Third, the chemiluminescences of the emitting antibiotics disappeared very quickly, the 
typical full width at half maximum (FWHM) was 2 sec (Figure 5-6); the slowest 
emission, from ampicillin, had a FWHM of 7 sec (Figure 5-7) and probably a diode-array 
detector is needed to observe the frill spectrum. 

However, there are still several questions remaining to be answered. 

1. When penicillin G was dissolved in an aprotic solvent like CH3CN, even after 
oxidation by the KO2 saturated solution, no chemiluminescent signal was observed. In 
dry CH3CN, KO2 exists in the form of superoxide anion radical. This may indicate that 
without dismutation reactions with water or other protic solvents, superoxide can not be 
transferred into the reactive species, and so the question arises what is the reactive species 
generated from the superoxide dismutation reactions? Since H2O2 is not able to generate 
chemiluminescence after oxidizing any of the p-lactam antibiotics, a possible candidate 
of this reactive specie is 'Oj. Further experiments are conducteded to prove its existence. 

2. Why do the six antibiotics including azetidinone, sulbactam, clavulanic acid, 
methicillin, penicillin V, and cefotaxime not produce chemiluminescence in the reaction? 
Why do the other seven antibiotics gave chemiluminescences of different intensities? 
What are the chemiluminescent intermediates and through which pathways do they go? 



57 



3. Is it possible that in this reaction the hydroxyl radical has been generated, i.e. is 
the oxygen toxicity theory based on the Haber- Weiss reaction the right assumption? 
To answer these questions, the degradation products of P-lactam antibiotics in the 
reactions with KOj needed to be identified, and a DO2 experiment needed be designed to 
prove the existence of 'O2 in the superoxide dismutation reaction. 

■■■'.''-'- '■ ■ ,V if. \ " 1^ 



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58 



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CHAPTER 6 
MECHANISTIC STUDIES 

Penicillin G was chosen as an example to investigate the mechanism of the 
chemiluminescence of P-lactam antibiotics interacting with potassium superoxide. 
Various experiments were conducted on the aqueous penicilhn G sample and the sample 
containing the products generated by oxidizing penicillin G with potassium superoxide. 
These experiments include nuclear magnetic resonance (NMR) assay, thin-layer 
chromatography (TLC), and high performance liquid chromatography (HPLC) using a 
ultraviolet (UV) spectrophotometer, a photodiode array (PDA) detector, and a mass 
spectrometry (MS) detector. In all these experiments, solid KOjWas utilized instead of 
the saturated KOj reagent (K02-acetonitrile-18-crown-6-ether) in order to simplify the 
experiments. A DO2 experiment was also run to prove the generation of singlet oxygen in 
the suproxide dismutation reaction. , 

',. ' '^ »' V ! j" The Deuteration Experiment 

Stauff et al. [104, 105] proposed that the spontaneous disproportion of superoxide 
generating 'O^, rather than ground state oxygen CO^), as shown in equation (1). This 
proposal had received support from the calculations of Knoppenol [106]. 



65 



66 



Khan [94, 95] had also proposed that superoxide is a precursor of 'O2, although he 
believes this occurred by a direct electron transfer reaction [equation (2)]. 

Oj- ^ e- + 'O2 (2) 

Nilsson and Keams [98], however, based on experiments injecting DjO rather than H2O 
into the dry dimethyl sulfoxide (DMSO) containing KOj, rejected the suggestion that 
superoxide serves as a direct precursor of 'Oj. The major question remaining in their 
investigation is the fact that even though it can be generated from KO2 after injection of a 
small amount of D2O or H2O, 'O2 will be mostly surrounded by DMSO, not by D2O or 
H2O because both react with superoxide through the dismutation reaction. Comparing the 
30 usee lifetime of 'Oj in DMSO with its 20 usee in DjO and its 2 ^sec in H2O it is not 
surprising that no difference was observed. Also, whether the frace amounts of singlet 
oxygen produced in this system is enough to oxidize the 'O2 acceptor tetramethylene 
(TME) is uncertain, and thus it is possible that no oxidation signal of TME could be 
detected in the KOj system adopted by Khan's experiments. 

Since a relatively strong chemiluminescence had been obtained in the reaction of 
aqueous penicillin G interacting with KO2, D2O was used instead of H2O to dissolve 
penicillin G, and to examine the deuterium effect on the chemiluminescent intensities. In 
view of the fact that in DjO 'O2 has more time to react with penicillin G, an enhancement 
of chemiluminescence supports the concept that the 'Oj is generated in the superoxide 
dismutation reaction. 4.0 mg/ml of penicillin G solutions were prepared in pure DjO, 
70% D2O-30% H2O, 50% D2O- 50% H2O, and pure H2O. The chemiluminescences of 2 



67 



ml of these solutions were tested by injection of 50 (jl of KOj, which is contained in a 
saturated solution made of 18-crwon-6-ether and acetonitrile. The experimental 
parameters were set as the following: Gain (PMT/HV) is 8.8, Sensitivity is set at auto, 
Time Constant is 5, and Suppression Background is set at high. The differently 
intensified chemiluminescences are described in Table 6-1, and the chemiluminescent 
profiles of penicillin G in the mixture solvents with varied DjO to HjO ratios (fi-om pure 
DjO-the top to pure HjO-the bottom) are demonstrated in Figure 6-1. Inspection of Table 
6-1 clearly shows an approximately 9-fold enhancement of chemiluminescent emission of 
penicillin G by replacing Rfi with DjO as the solvent. Also lowering the ratio of D^O to 
HjO was correlated with decreased intensities of chemiluminescences. Another 
observation really attracts attention. When the high voltage of photomultiplier tube 
(PMT) was adjusted to 980 volts, the time constant was set at 0.25 to catch the weak and 
short-life emissions. After the first injection of KO2 into the solution of penicillin G in 
pure DjO, the huge numbers of photoelectrons collected by the PMT outscaled the 
detector. Characteristically, even after a second, or third injection of KOj into the same 
reacting cell, a significant signal was still observed, although their intensities dramatically 
decreased. This is totally different from the case of the penicillin G in pure HjO, where a 
much weaker chemiluminescence from the first injection of KOj was detectable, but no 
signal was observed after the second injection of KOj. This phenomenon fiirther suggests 
that 'O2 can be generated in the superoxide dismutation reaction. In this detection system, 
the syringe needle was connected to a long, curved metal tube, which is immersed in the 



68 



center of the cell to prevent the light leakage caused by injection. After injection, KO2 
diffuses radially from the center to the cell wall. Most penicillin G reacts with the KO2 
and the concentration of penicillin G is decreased, especially in the center. KO2 from the 
second or third injection has to fravel relatively longer distance to react with the 
remaining penicillin G. The singlet oxygens produced from the second or third injection 
of KO2 into the D2O therefore have more opportunity to react than those in H2O because 
of the longer lifetime of 'O2 in D2O. In the contrast, 'O2 formed in H2O can not fravel far 
enough to react with penicillin G away from the center. The repeated injections is an 
advantage of investigations in the static mode. 

The results presented in this experiment sfrongly reinforce that in the contrast of 
the Haber- Weiss reaction, the 'Oj can be generated by superoxide dismutation reaction, 
which can occur either spontaneously or when catalyzed by SOD with great rate. It 
indicates that superoxide maybe must not have time to fiuther react with hydrogen 
peroxide to produce hydroxyl radicals and cause oxygen toxicity, as described by the 
Haber- Weiss reaction. Even though it is catalyzed by fransition metals, which are 
available in biological systems, the Haber- Weiss reaction will face competition from the 
Fenton type reaction [equation (3), (4)]. 

O2- + Fei° ^ Fe" + O2 (3) 

Fe° + H2O2 ^ Fe°' + OH + OH' (4) 



69 



Equation (4) is consistent with the fact that plenty of H2O2 has been generated ahnost 
instantly by the superoxide dismutation reaction. It is already claimed that the evidence 
for the formation of OH is overwhehning [107, 108,109, 110]. This does not, of course, 
preclude the formation of reactive oxygen species in additional to OH, for example 'O2 in 
equation (3). Taking account of the fact that hydroxyl radicals may also come from the 
Fenton type reaction as well as the Haber- Weiss reaction, and the 'O2 is capable of 
damaging the same biological components as is superoxide, not all the oxygen toxicity 
should be rationalized by the Haber-Weiss reaction. The toxic species should be specified 
in order to better understand the pathology, which causes the diseases, and to develop 
new therapeutic strategies as well as new antioxidants. In the experiments discussed 
above, it is strongly suggested that 'O2 well deserves more attention, and it is probably 
the prime oxidant in various diseases caused by oxygen toxicity. 

NMR Consideration 

Two sample of 1 5 mg of penicilhn G ( or penicillin V) in 0.75 ml deuterium oxide 
were made, one of them was reacted with 10 mg KO2. The 300 MHz 'H-NMR, 75 MHz 
"C-NMR and Attached Proton Test (APT) were obtained by a GEMINI-300 NMR 
spectrometer at room temperature. 3-(Trimethylsilyl) propionic-2, 2, 3, 3-d4 acid, sodium 
salt (TSP, Aldrich) was used as internal NMR standard and the HDO peak appeared 
around 4.8 ppm from TSP. These NMR assays are used to detect the structural alternation 



70 



before and after the oxidation of penicillin G or penicillin V by KOj and to check the 
purity of these two drugs. 
NMR Spectra of PenicilHn G 

Figure 6-2 is the proton NMR spectrum of potassium penicillin G in Dfi. The 
interpretation of the spectrum is given below. 



^^^f Chemical Shift (ppm) Position 

^^9\/"^— « O 1.501 (s), 1.570 (s) 1,2 

^^3C s^^-^\ // 3.615, 3.630 (ABq,J= 14 Hz) 10 

HN--^ 4.247 (s) 4 

1° 5.430 (d, J=3.9 Hz) 6,7 

peniciUinG f^\ 5.493 (d, J-3.9 Hz) 6,7 

4, f 7.278-7.392 (m) 12,13,14 



Figure 6-3 depicts the '^C-NMR spectrum of potassium penicillin G in DjO. The 

assignments are tabulated below. 

. ;;• .i ......?..■ •^'-. H 

' .* Chemical Shift (ppm) '•' '-J Positbn 

26.594,30.843 ' 1,2 

42.088 lb 

58.190 7 

64.518 3 

66.779 6 

73.305 4 

127.619 14 

129.137,129.486 12,13 

134.600 ll' 

174.345, 174.497, 174.786 5, 8, 9 



71 



The results here of 'H-NMR and "C-NMR spectra are similar to these presented 
in a study of penicillins and cephalosporins [111]. 

Compared with Distortionless Enhancement by Polarization Transfer (DEPT), 
APT is less sensitive and does not distinguish between CH3 and CH peaks (both down) or 
between CHj and quaternary C peaks (both up). However, in the case of penicillin G and 
penicillin V, the partial information obtained by APT is sufficient for interpretations. As 
shown in Figure 6-4, the CH3 peaks at position 1, 2 and CH peaks at position 4, 6, 7, 12, 
13, 14 are down. Since the stronger peaks at 127.619 ppm, 129.137 ppm, and 129.486 
ppm obviously belong to the aromatic carbons, the other down peaks are easily identified. 
For the up peaks, the peaks at 174.786 ppm, 174.497 ppm, and 174.345 ppm are clearly 
from carbonyl groups; the peaks at 134.600 ppm and 64.5 18 ppm refer to the quaternary 
carbon of aromatic ring and alkanes respectively. Subsequently the peak at 42.008 ppm is 
identified as CH2 at position 10. 

The degradation products of penicillin G generated by reaction with potassium 
superoxide were also examined by NMR spectroscopy without isolation as shown in 
Figure 6-5 ('H-NMR spectrum), and Figure 6-6 ('^C-NMR spectrum). The APT spectra 
of degradation products were not readable and so are not presented. Interestingly, the 
NMR signal at position 10 in Figure 6-6 seems to have disappeared. Probably penicillin 
G lost the CH2 at position 10 after oxidation by KO^. However, there is a peak at 42.589 
ppm close to 42.088 ppm (position 10 in Figure 6-3) in the '^C-NMR spectrum, which 
may suggest that the electronic environment has changed but CH2 still exists. More 



72 



experiments are needed to clarify the status of the methylene, which is between the 
carbonyl and benzyl moiety on penicillin G. 

Meanwhile the comparison between the NMR spectra of the phenyl ring before 
and after the oxidation by KO2 draws attention. If the hydroxyl radical is generated by the 
interaction between superoxide and hydrogen peroxide, which is produced by the 
superoxide dismutation reaction, the hydroxylate aromatic compounds should be 
identified by NMR spectra since the electrophilic hydroxyl radical adds readily to the 
aromatic nucleus (K = 3 ~ 8 x lO'M" S') [112]. No such hydroxylate compounds are 
demonstrated in Figure 6-5 and Figure 6-6. There is no disubstitution or chemical shift of 
the aromatic ring to suggest the hydroxylation. Hence, no conclusion can be drawn that 
the hydroxyl radical is formed by the Haber- Weiss reaction in this superoxide system. 
NMR Spectra of Penicillin V 

As shown in Figure 5-1, penicillin G emitted the strongest chemiluminescence 
after the oxidation by KO2 and surprisingly no chemiluminescent signal was observed 
with penicillin V under the same reacting conditions. Considering the fact that the only 
structural difference between penicillin G and penicillin V is the "phenoxyl methyl" 
moiety of penicillin V, the oxidation of potassium penicillin V in D2O was examined by 
NMR spectroscopy by comparing the spectra following the oxidation of penicillin G and 
penicillin V by KOj. 

The proton NMR spectrum of penicillin V in D^O is shown in Figure 6-7, and the 
interpretation is described as below. 



73 






/ 



/ 



X 



0==\ 



OH 



./T 



10' 



— O 



\ 



\ 



11=12 
14 




penicillin V 



Chemical Shift (ppm) Position 



1.500,1.513(8) 


u 


4.260 (s) 


4 


4.469 (ABq, J=16.2 Hz) 


10 


5.520 (d, J=4.2 Hz) 


6,7 


5.569 (d, J=3.9 Hz) 


6,7 


6.837-7.273 (m) 


12,13,14 



Figure 6-8 is the carbon NMR of penicillin V in DjO. The peaks are assigned as 
the followings, which are in agreement with the literature values [113]. 



Chemical Shift (ppm) 

26.548,31.238 

57.492 

64.670 

66.49 P 

66.613" 

73.260 

114.841 

122.384 

130.002 

156.908 

170.643 

174.285" 



Position 

1,2 
7 
10 
3 

4 
12 
13 
14 
11 
5 
A9 



"the peaks with chemical shifts of 66.491 and 
66.613 ppm are overlapping and they are 
differ enciated by APT (Figure 6-9). 
"the peaks of carbons at position 8 and 9 are 
overlapping at 174.285 ppm. 



74 



In case of penicillin V, APT is very useful to identify carbon 3 at 66.492 ppm (up) 
and carbon 6 at 66.597 ppm (down) as shown in Figure 6-9. The degradation products of 
penicillin V after the oxidation by KO2 were also studied by proton NMR (Figure 6-10) 
and carbon NMR (Figure 6-1 1). Both showed no structure alternation on the aromatic 
ring, and contrary to penicillin G the CHj at position 10 remains intact. 
Conclusion 

As discussed in chapter 1 (equation 7), the quantum yield of chemiluminescence 
is associated with Or, the yield of product; O^s.the number of molecules entering the 
exited states; and Op the fluorescence quantum yield. Therefore, the generation of 
chemiluminescent products is the first step to emit chemiluminescence. It seems that 
unlike penicillin G, penicillin V did not carry out this first step and no chemiluminescent 
signal was observed. The NMR data here suggests that the CH2 at position 10 be 
correlated with the chemiluminescent emissions, and no hydroxylate aromatic compound 
was found to support the possibility that hydroxyl radical was generated by the Haber- 
Weiss reaction in the current superoxide system. 

TLC Analysis 

Introduction 

The TLC procedures have been used to allow the simple and rapid separation and 
identification of the different (spontaneous, chemical and enzymatic) degradation 
products of penicillins and cephalosporins [114]. After testing several spray reagents, Lin 



75 



and Kondo [115] found that the iodine-azide reagent was the best for detection of 
penicillin G, producing a yellow color on the TLC plate; the vanillin-phosphoric acid 
mixture was the best for the detection of streptomycin (brown) and dihydrostreptomycin 
(brown); ninhydrin was best for kanamycin (purple) and fradiomycin (purple). Since the 
NMR spectra had indicated that numerous degradation products of penicillin G were 
generated after the oxidation by KOj (Figure 6-5 and 6-6), these three reagents (iodine- 
azide, vanillin-phosphoric acid, and ninhydrin) were utilized in the TLC analysis to 
separate the hydrolysis products from the oxidation products or other derivatives of 
penicillin G. It is anticipated that sufficient samples would be obtained to conduct 
experiments using mass spectrometry after separation and purification. 
Experimental 

TLC plates (1 inch x 3 inches) of silica gel were used for separation. Five 
different solvent systems were tested: (A) n-butanol-water-ethanol-acetic acid 
(5:2:1.5:1.5); (B) n-butanol-water-acetic acid (4:1:1); (C) acetone-acetic acid (19:1); (D) 
acetone-water (17:1); (E) methanol-water (50:50). Among them, solvent system (B) was 
the best for separating the degradation products of penicillin G. Each TLC plate was 
sprayed consecutively with the following spray reagents: (a) ninhydrin, (b) iodine-azide, 
(c) vanillin-phosphoric acid. The iodine-azide reagent was prepared by dissolving 0.5076 
g iodine (Sigma) and 58.9 mg sodium azide (Fisher) into 200 ml chloroform to make a 
solution of 0.01 N iodine-0.02 % azide. Vanillin (Sigma) 0.9992 g was dissolved in 100 
ml phosphoric acid and this solution was diluted 1:1 with methanol to form the vanillin- 



76 



phosphoric acid reagent. Ninhydrin 0.2010 g was dissolved in 5 ml of 10 % acetic acid, 
and added to 95 ml of n-butanol to produce the ninhydrin reagent. Since spraying 
vanillin-phosphoric acid mixture did not mark any new spot, as shown in Table 6-2, this 
reagent was not used in the separation procedures. 
Results and Discussions 

Each of the aqueous penicillin G sample (sample PG) 10 mg/ml and the sample 
containing degradation products of penicillin G (sample PGKOj) was made as in the 
NMR experiments. Both of them were adjusted to pH 12.00 and were spotted on the same 
TLC plate. Each spot was dried with pressured air and the spotting was repeated four 
times to increase the amount of sample in each spot. After the first elution in the solvent 
system (B), the TLC plate was dried by a hot plate, then ninhydrin and iodine-azide were 
sprayed consecutively on the TLC plate. Three spots were found originating from sample 
PGKO2, two spots from sample PG. The colors and the moving distances of these spots 
were shown in Figure 6-12. Obviously the second spot from sample PGKO^ and the first 
spot from sample PG have the same Revalue (distance solute moves/distance solvent 
front moves). After the third elution, three spots of sample PGKO^ were found to have the 
same Revalues as those of sample PG, as shown in Figure 6-12. This indicates that while 
it was oxidized by KO^, the aqueous penicillin G was hydrolyzed as well because of the 
sfrong basicity of superoxide in water [116]. Therefore, the first three spots of sample 
PGKO2 after the third elution were believed to be produced from the reactions other than 
hydrolysis. 



77 



To prepare for the mass spectroscopy experiments, 100 mg/ml of sample PGKOj 
was made and put on one of the 36 TLC plates in a line spots. Each spot was dried by 
pressured air and the spotting was repeated four times. The plate was sprayed after the 
third elution, 4/5 of the plate was covered by a piece of paper and the other 1/5 was 
sprayed by ninhydrin and iodine-azide, as shown m Figure 6-13. The invisible three lines 
on the 4/5 part of the TLC plate was located by the sprayed 1/5 part of the plate, then 
each line on the 4/5 part was cut off separately. After the three desired block on all the 36 
plates were cut off and separated into three samples, each of them was washed by 
methanol and the silica gel was filtered. The subsequent solution was concentrated into 
three approximate 1.5 ml solution (solution 1, 2, and 3). Each solution was repeatedly 
spotted 12 times on a TLC plate (Figure 6-13) and developed in the solvent system (B). 
After the first elution and the two reagents were sprayed, two spots were found from 
solution 1, one from solution 2, and none from solution 3. Possibly solution 3 results 
from the running solvents or from the solvent of sample PGKO2, as its spots were 
overlapping with the solvent front. These four spots were named 1 1, 12, 13, and 3. 
Following the same procedures of mass production as discussed before, four solid sample 
were finally obtained after filtering, and drying and were analyzed by Fast Atom 
Bombardment (FAB)-mass specfroscopy. The average lengths of the time consumed 
during each run are listed in Table 6-3. 

One peak of m/z 105 with quite low intensity was found in one of the four 
samples, as shown in Figure 6-14. This is most likely ph-C=0, however, in the other 



78 



three sample spectra it appears that they was enough sodium salts in the samples to 
suppress other signals. The sodium salts may come from the TLC plates or from other 
reagents. Although it is consistent with the results of NMR assay, in which the CHj seems 
lost after the aqueous penicillin G was oxidized by K02, the peak m/z 105 in mass 
spectrum of spot 13 seems unreliable and better separation methods are needed. 

HPLC Detection 

t, 

HPLC is widely utilized to analyze the degradation products of penicillin G in 
aqueous solutions because of its specificity, ease of use and high sample capacity. 
However, no product generated by the oxidation of penicillin G had been studied by 
HPLC. In this section the products of aqueous penicillin G following the oxidation by 
potassium superoxide is investigated by HPLC using a CI 8 column. 
Experimental 

The HPLC experiments are conducted with the following equipment: 

1) Beckman Model 11 OA pump 
Beckman Model 153 UV detector 
Beckman Instrument Inc., FuUerton, CA. 

2) Hewlett-Packard 3392A integrator 
Hewlett-Packard Co., Palo Alto, CA. 

3) Waters 501 HPLC pump 

Waters™ 1 996 Photodiode Array Detector 



79 



Waters™ 7 1 7 plus Autosampler 
Millipore Corporation, Milford, MA. 

4) Exsif ODS column, 150 by 4.6 mm dimension, 7 |im particle size, 100 A pore size. 
Keystone Scientific, Inc., Bellefonte, PA. 

5) Helium compressed gas, Gainesville Welding Supply, Gainesville, FL. 

Various reference compounds were injected and the retention times were 
measured in order to identify the unknown peaks, which were produced by the 
degradation of penicillin G following the oxidation by potassimn superoxide. Hydrogen 
peroxide was obtained from Fisher. 3,4-dihydroxybenzoic acid, 2, 4-dihydroxybenzoic 
acid, 2, 3-dihydroxybenzoic acid, phenylacetic acid, p-hydroxyphenyl acetic acid, and 
benzaldehyde were bought from Aldrich. Benzoic acid was obtained from Sigma. 
P-hydroxybenzoic acid was obtained from Eastman (Eastman Organic Chemicals, 
Rochester, NY). Salicylic acid was obtained from Mallinckroot (Mallinckroot Chemical 
Works, St. Lois, New York, and Montreal). All the chemicals, including the reference 
compounds and the samples, were completely dissolved using an ulfrasonic cleaner. All 
the reagents utilized by the HPLC system were filtered and degassed for about 10 
minutes. Prior to the experiments, the HPLC setup was washed by water, 50:50 
watenmethanol, and methanol consecutively, each for half-hour. The UV detector was set 
at 254 nm, 0.08 absorbance unit fiiU scale. The flow rate was 1.0 ml/min and the pump 
pressure was kept below 4000 psi. The 2 mg/ml of sample PG and PGKOj were prepared 
as in chapter 3. 



80 



Results and Discussion 

30% MeOH-70% phosphate buffer (pH4.15) is good for the separation of the 
hydrolysis products of penicillin G in aqueous solutions, but is not good for oxidized 
sample, which is consistent with the observation of Vadino et al. [117]. Acetonitrile 0.1% 
in pH 5.66 phosphate buffer was found useful for the detection of the oxidation products 
of penicillin G as shown in Figure 6-15. Unfortunately, none of the retention times of 
unknown peaks of sample PGKOj matched up with that of the reference compounds as 
shown in table 6-4. Since the same CI 8 column was not available, the experiments using 
photodiode array detector (PDA) were conducted on a different Exil'^ODS column. 
Similar results were obtained as shown in Table 6-4. 
Conclusion 

1 . When the sample PGKOj stayed overnight huge peaks appeared hours after the 
injections. Were polymers generated? 

2. No benzoic acid or its derivatives were identified by this kind of detection and maybe 
the amount generated is so small that it is out of the detecting limit of the UV 
detector. '"'■. ' '"'■' ' 

3. UV detector is not suitable for the detection of the mixture caused by oxidation and 
hydrolysis. Mass spectrometer is the obvious choice. 

4. The HPLC buffer of sodium salts must be replaced by ammonium buffers or other 
nonmetal-ion solutions since in the TLC analysis vast amounts of sodium salts 
suppress the signal for mass spectroscopy. 



81 



HPLC/ES I-MS and HPT C/APCI-MS Analysis of the nemdation of Penir.illin O 
Following the O xidation hv Potassium Sup ernyidt^ 

Introduction 

Electrospray ionization mass spectrometry (ESI-MS) was first introduced by 
Yamashita and Fenn [118, 119] in 1984. At approximately the same time, a veiy similar, 
independent development was reported by Aleksandrov and coworkers [120]. Actually, 
electrospray as a source of gas-phased ions and their analysis by mass spectrometry was 
proposed much early by Dole [121], however Dole's experiments were too narrowly 
focused on the detection of polymeric species, which are not themselves ionized in 
solution, and the experimental results were not convincing [122]. 

Since the end of 1980s, ESI-MS has developed at a tremendous pace and 
established itself as an outstanding method for biochemical applications. It has permitted 
new possibilities for mass spectrometric analysis of high-molecular-weight compounds of 
all types, including proteins, nucleotides, and synthetic polymers. Other analytical 
techniques do not provide the same level of detailed information regarding molecular 
weights and structures from extremely small amounts of material. Compared to the other 
mass spectrometric ionization techniques, ESI has three superior features. First, ESI has 
the truly unique ability to produce extensively multiply charged ions, which may be 
analyzed on virtually all types of mass spectrometers. Second, the samples analyzed by 
ESI-MS must be introduced as a liquid phase. This results in a natural compatibility of 



82 



ESI with many types of separation techniques. Third, the extreme "softness" of ESI 
process allows the preservation in the gas phase of noncovalent interaction between 
molecules as well as the study of three-dimensional conformation. For earlier ionization 
methods such as fast atom bombardment (FAB) and plasma desorption, abundant energy 
is applied in a highly localized fashion over a short time. These methods lead not only to 
ion desolvation but also to fragmentation or even net ionization, that is , the creation of 
ions from neufrals. As for ESI-MS, in which the desolvation is achieved gradually by 
thermal energy at a relatively low temperature as it is a far softer technique. 

There are three major steps in the production of gas-phase ions from electrolyte 
ions in solution by elecfrospray: (1) production of the charged droplets at the ES capillary 
tip; (2) shrinkage of the charged droplets by solvent evaporation and repeated droplet 
disintegrations, leading ultimately to very small highly charged droplets capable of 
producing gas-phase ions; and (3) the actual mechanism by which gas-phase ions are 
produced from the very small and highly charged droplets. Kebarle and Tang [123] 
described the first two steps in Figure 6-16. As shown in this schematic representation of 
the charged-droplet formation, a high voltage of 2-3 KV is supplied to the metal capillary 
which are typically 0.2 mm o.d. and 0.1 mm i.d. and located 1-3 cm from the counter- 
electrode. Penetration of an imposed electric field into the liquid leads to formafion of an 
electric double layer in liquid. Enrichment of the surface of the liquid by positive 
elecfrolyte ions leads to destabilization of the meniscus and formation of cone and jet 



83 



emitting droplets with excess of positive ions. Charged droplets shrink by evaporation 
and spht into smaller droplets and finally gas-phase ions. 

More recently, HPLC/ESI-MS has gained widespread recognition as a powerful 
analytical tools for drug metabolism and pharmacokinetic studies because it permits the 
separation and ionization of polar nonvolatile or thermally labile compounds, such as 
drugs, conjugated metabolites, peptides or DNA adducts. In addition, these techniques 
facilitate qualitative and quantitative studies on the parent compounds and their polar 
metabolites. Given its attractive advantages such as good sensitivity, reliability, and 
specificity for a wide variety of compounds with minimal sample handling, HPLC-MS 
has replaced GC-MS, which requires sample derivatization for polar metabolites. 

A series of ionization sources compatible with HPLC is available, which include 
thermospray ionization (TSP), electron impact (EI) or chemical ionization (CI) with 
particle beam interface (PB), continuous-flow fast atom ionization (CFFAB) and 
atmospheric pressure ionization (API) using a heated nebulizer interface (APCI). Most of 
those methods have their limitations. TSP and APCI require critical control of the 
vaporizer temperature during analysis, and thermal degradation of labile molecules may 
occur. CFFAB can only accept low flow rates, and requires the presence of a matrix to 
assist ionization, thereby affecting the ion-current stability and making the method 
susceptible to interference from background ions. However, HPLC/ESI-MS is now fi-ee 
of most major technical problems and has become the method of choice for drug 
metabolism and pharmacokinetic research, as reflected in the impressive number of 



84 



applications published in the last few years. More encouragingly, a triple-quadrupole 
mass spectrometer is capable of performing MS/MS such as neutral-loss scan, parent-ion 
scan or product-ion scan. Therefore, besides the molecular weight, HPLC/ESI-MS/MS 
(or MS") can give complex mixture analysis, structural elucidation, combinatorial 
screening, fast chromatography, picogram quantitation and protein sequencing. For 
example, in addition to MS/MS capabilities, the LCQ LC/ MS" Detector (octapole- 
octapole mass spectrometer, Finnigan MAT, San Jose, CA) also provides standard 
MS/MS/MS. . .. MS" (n=l-10) for structural elucidation capability. The selectivity of MS" 
means that a compound can be fragmented, and the resulting fragments fiirther isolated 
and analyzed to yield structural information about complex molecules. The results 
presented here confirm that HPLC/ESI-MS/MS is an outstanding tool for pharmaceutical 
analysis. 
Experimental 

AH data were obtained on a Finnigan MAT (San Jose, CA) Benchtop LCQ 
LC/MS" Detector (external ionization ion trap) equipped with both an electrospray 
ionization (ESI) ion source and an atmospheric pressure chemical ionization (APCI) ion 
source. These sources were interfaced to a Hewlett-Packard (Palo Alto, CA) 1090 (Series 
II) high pressure liquid chromatograph (HPLC). The effluent from the HPLC column was 
typically split prior to the ion source. 

Chromatographic separation was achieved on a Keystone Scientific (Bellefonte, 
PA) Exil"^ ODS (C-18; 150x4.6 mm dimension; 7 ^m particle size; 100 A pore size) 



85 



HPLC column utilizing a reverse phase gradient. Two mobile phases were used. Mobile 
phase A was 0.1% acetic acid in 50:50 water: acetonitrile and mobile phase B was 0.01 M 
ammonium acetic acid (pH 5.72) with 0.1% acetonitrile in water. The flow rate was 1 
ml/min and the loop size is 5 jil. After 1 mg/ml and 0.1 mg/ml penicillin G were oxidized 
respectively by solid KO2, the degradation of penicillin G was analyzed via direct 
infusion between HPLC and ESI-MS at room temperature. A number of different 
gradients were utilized as described in table 6-5. 
Results and Discussion 

A number of derivatives and their isomers of penicillin G were separated and 
identified by HPLC/ESI-MS/MS after the interaction between aqueous penicillin G and 
solid potassium superoxide. Most of these can be classified into the products of 
hydrolysis, oxidation, recombination reactions including dimerization, and 
chemiluminescent reaction. 

Among these compounds benzylpenilloic acid (MW 308), benzylpenicillenic acid 
(MW 334), benzylpenillic acid (MW 334), and benzylpenicilloic acid (MW 352) were 
found which arise fi-om the base-catalyzed hydrolysis of penicillin G in aqueous 
solutions. The chromatograms and mass spectra of HPLC/ESI-MS/MS of these four 
compounds plus penicillin G and the interpretations of mass spectra are presented in 
appendix A. The existence of these compounds was supported by the degradation 
pathway of penicillin G proposed by Kessler et al [124]. 



86 



The oxidation of penicillin G and its derivatives occurred exclusively on the 
sulfur atom. Derivatives like compound 312 (a product of the oxidation of penicillamine, 
which is one of the products of hydrolysis of penicillin G), bertzylpenilloic acid sulfoxide 
(MW 324), benzylpenillic acid sulfoxide (MW 350), and benzylpenicilloic acid (MW 
368) are examples. Two isomers of penicillin G sulfone were also found and the 
experimental results and their interpretations are recorded in appendix B. 

The recombination of the derivatives of penicillin G resulted in several new 
compounds, such as the dimer of penicillamine (MW 296), compound 440 (a product of 
recombining of compound 269 and benzylpenilloaldehyde, one of the hydrolysis products 
of penicillin G), compound 632 (a product of penicillamine dimer and ben2ylpemcilloic 
acid), and compound 642 (a product of benzylpenilloic acid and benzylpenicillenic acid). 
The HPLC/ESI-MS results and identifications of these compounds are in appendix C. 
The structures of these compounds and the mechanism of dimerization are simunarized in 
table 6-6. It is believed that the thiol (penicillamine) loses a proton, and then it is oxidized 
by oxygen to form a free radical and radical coupling generates the dimer [125]. Singlet 
oxygen is probably involved in this process. 

The identification of benzoic acid, compound 336, and compound 511 (a product 
of compound 336 and benzylpenilloaldehyde) appears to support that a chemiluminescent 
reaction was involved in the degradation of penicillin G following the oxidation by 
potassium superoxide. The data of mass spectra of these three compounds and their 
interpretation are shown in appendix D. 



87 



In addition, compound 269 and compound 353 were identified by their mass 
spectra, as described in appendix E, but their sources are unknown. 

Figure 6-17 illustrates the total chromatogram between 0-26 min of HPLC/ESI- 
MS of sample PG and sample PGKOj while the HPLC gradient is 100% mobile phase B / 
0% mobile phase A. The top chromatogram demonstrates the hydrolysis of penicillin G 
in the aqueous solution. The peak with the retention time of 22.15 min is most likely 
penicillin G and the other major peaks at 19.94 min, 20.08 min, 20.91 min, 22.59 min are 
the derivatives of penicillin G after hydrolysis. All of these were identified by their ESI- 
MS/MS and verified in the gradient, 100% mobile phase A / 0% mobile phase B. The 
retention time in different gradients, the structures, the molecular weight, and the mass / 
charge ratios of the parent ions are listed in table 6-7. The bottom chromatogram was 
acquired from sample PGKOj and it clearly shows that during the process of oxidation of 
penicillin G by potassium superoxide, the hydrolysis of penicillin G occurs as well. This 
is indicated by the observation that the intensities of the peaks at 19.88 min (the 
overlapping of peaks at 19.94 min and at 20.08 min in the chromatogram of sample PG) 
and at 22.59 min increased significantly, however, the peak of penicillin G at 22.15 min 
seems to have disappeared in the chromatogram of sample PGKOj. The experimental 
results are consistent with the fact that superoxide is a strong base in aqueous solution 
[1 16] and once it entered aqueous solutions, it becomes a catalyst for the hydrolysis of 
penicillin G; thus, the degradation rate was increased. A close-look at the chromatogram 
of sample PGKOj, as shown in Figure 6-18, shows that there are two major peaks with 



88 



retention times at 20.56 min and 20.81 min, which have no equivalents in the 
chromatogram of sample PG. The interpretation of the HPLC/ESI-MS of those two peaks 
suggests that they are ben2ylpenicillic acid sulfoxide and benzylpenilloic acid sulfoxide, 
as shown in table 6-8. Actually, by using different HPLC gradients, numerous sulfoxide 
of the derivatives of penicillin G were found, which were summarized in Figure 6-19, and 
the pathways of oxidation are specified. 

Consider the chemiluminescences of thirteen p-lactam antibiotics. Since there are 
some sulfur compounds like Martin's sulfiirane, which becomes chemiluminescent 
through sulfoxide or sulfone in the presence of DBA (9, 10-dibromoanthracene) [126], or 
rubrene [127], perhaps sulfoxide is the right intermediate to give chemiluminescence 
from P-lactam antibiotics. This is consistent with the fact that no chemiluminscence was 
observed with the reactions with azetidinone, clavulanic acid, and sulbactam. They either 
have no sulfur, or the sulfur is aheady oxidized to sulfone, as shown in table 6-9. This 
concept is not supported by the fact that as shown in table 6-9, methicillin, penicillin V, 
and cefotaxime did not emit chemiluminescence, even though they have sulfur in their 
structures. It is also impossible to rationalize the differently intensified 
chemiluminescences of penicillin G, dicloxacillin, piperacillin, ampicillin, cephalothin, 
amoxicillin, and hetacillin in table 6-10 when such a proposal is adopted. Furthermore, 
the maximum wavelength of chemiluminescence emitted from SO2 ranges from 260 nm 
to 480 nm; for S^, it is 275-425 nm; for HSO, it is 360-380 nm (table 1-4). Obviously, all 
of them don't match with the 535 nm of penicillin G, as determined in chapter 5. 



89 



Therefore, the claim that the chemiluminescence emitted by p-lactam antibiotics came 
from the oxidation of sulfur is not supported. On the other hand, the existence of benzoic 
acid, compound 336, and compound 511 in the degradation of penicillin G was proved by 
their HPLC/ESI (or APCI)-MS/MS and the involvement of singlet oxygen in the 
chemiluminescent reaction of the penicillin G reaction was verified by the DjO 
experiments. It is proposed that the autoxidation initiated by singlet oxygen is the 
mechanism of the chemiluminescences emitted by P-lactam antibiotics. As shown in 
Figure 6-20, the dismutation of potassium superoxide generates singlet oxygen ('Oj) and 
as a strong base in aqueous solutions, superoxide facilitates the removing the a-proton by 
'Oj to form dioxetanone and 6-aminopenicillanic acid (6-APA). Dioxetanone then loses 
carbon dioxide and generates the excited phenylcarbonyl cation, which emits the 
chemiluminescence at 535 nm by relaxation. Phenylcarbonyl cation can either recombine 
with 6-APA to produce compound 336, or be ftirther oxidized by 'O^ (or H^Oj) to 
produce benzoic acid. Compound 51 1 is formed when compound 366 reacts with 
benzylpenilloaldehyde. Compound 336 and compound 511 are found by electrospray 
ionization (ESI) mass spectrometry. The mass spectra of individual peaks are also 
obtained to further confirm the structures (MS/MS). The presence of benzoic acid has 
been proved by negative atmosphere pressure chemical ionization (APCI) mass 
spectrometry. This type of reaction has two characteristic features: (1) an acidic a-proton 
whose removal by 'O2 leaves a special stabilized carbon radical, (2) a good leaving group 
expelled by the attack by peroxide to give a dioxetanone. This proposed mechanism is 



90 



applied to rationalize the observations of table 6-9 and table 6-10. In table 6-9, the lack of 
side chains on azetidinone, sulbactam, and clavulanic acid results in the lack of 
chemiluminescence; no a-proton on methicillin and cefotaxime results in no 
chemilimiinescence; for penicillin V, the oxygen on the side chain will repels the attack 
of 'O2 on an a-proton and the chemiluminescence is inhibited. In table 6-10, although 
penicillin G emits the most intensified chemiluminescence, its chemiluminescent 
intensity is far lower than luminol, lucigene, luciferin, and other chemiluminescent 
compound. The conformation of penicillin G determines that the carbonyl and benzyl 
moiety can not be on the same plane, and the lack of conjugated electron-delocalized 
structure leads to decreased electron withdrawing ability, thus, the removal of the a- 
proton is restricted. The variation of the acidity of a-protons also indicates that the 
chemiluminescent intensities of amoxicillin < ampicillin < piperacillin < penicillin G, 
which is consistent with their decreased electron-withdrawing abilities. Because 'O2 can 
works as an oxidizer for the sulfur of cephalothin and as a base to hydrolyze the side 
chain of hetacillin, whose five-member-ring side chain makes it more susceptible to 
hydrolysis than that of the other antibiotics, it is not surprising that the chances for 
chemiluminescence are decreased. The bulky side chain on the hetacillin can block the 
attack of 'O2 on a-proton, which combined with ring strain facilitate the hydrolysis of the 
side chain. In an attempt to explain the large chemiluminescent intensity obtained with 



91 



dicloxacillin, which does not have an a-proton, a particular mechanism is proposed in 
which the hght is emitted by the dioxetan as shown below: 




Extensive studies suggested that the most convenient method to produce 
dioxetans and dioxetanones is following reactions of olefins with singlet oxygen [US- 
IS 1]. Various dioxetans fall sharply into two classes. Dioxetans with alkyl aryl or other 
substituents ("simple dioxetans") with little electron donating power yield triple exited 
carbonyl products, whereas the second type, with strongly electron donating substituents 
(mainly nitrogen and oxygen) gives very high yields of singlet excitation [132]. Possibly, 
the dioxetan of dicloxacillin belongs to the second class and further investigations are 
needed. 

Finally, much data acquired fi-om the HPLC-MS, D^O, NMR, and TLC suggest 
that the mechanism of chemiluminescence of p-lactam antibiotics is autoxidation initiated 
by singlet oxygen. 



92 



Table 6-1: Effects of deuterium oxide on the intensities of chemiluminescence of 
penicillin G. 



Deuterium Oxide concn, % 



Relative integrated chemiluminescent 
intensity 



100 

70 

50 





204 
61 
22 
20 



Table 6-2: The response of spraying the three reagents on sample PGKO2 and sample 
KO,. 





PGKO2 


KO2 




color 


# of spots 


# of spots 


Vanillin 


brown 


1 , L=3.0cm 


none 


lodine-Azide 


yellow 


1 , L=3.0cm 


none 


Ninhydrin 


purple 


3, L=1. 3, 2.5, 3.2cm 


none 



Table 6-3: The elution time of TLC developments. 





Elution time (min.) 


Average elution 
time (min.) 




Plate 1 


Plate 2 


Plate 3 


Plate 4 


Plate 5 


Istelution 


41 


44 


42 


44 


44 


43 


2nd elution 


39 


39 


37 


40 


40 


39 


3rd elution 


40 


38 


37 


39 


36 


38 



93 



Table 6-4: The retention times of the reference compounds and sample PGKO2 



Injection 


Concentration 


Retention Time (min) 


Retention Time (min) 






with UV at 254 nm 


with PDA" 


hydrogen peroxide 


0.03% 


1.63 


1.692 


3, 4-dihydroxyl benzoic acid 


0.01 mg/ml 


2.71 


2.795 


p-hydroxyl benzoic acid 


0.01 mg/ml 


4.19 


4.192 


2, 4-dihydroxyl benzoic acid 


0.01 mg/ml 


4.20 


5.178 


2, 3-dihydroxyl benzoic acid 


0.01 mg/ml 


4.41 


5.033 


p-hydroxyl phenyl acetic acid 


0.01 mg/ml 


6.28 


6.805 


benzaldehyde 


1/250^ 


8.95 


8.907 


benzoic acid 


0.01 mg/ml 


9.00 


8.754 


phenyl acetic acid 


0.01 mg/ml 


13.60 


14.464 


salicylic acid 


0.01 mg/ml 


14.35 


14.97 


major peaks of sample PGKOj 


2.0 mg/ml 


3.61 


4.267 






14.40 


14.433 






21.99 


25.333 



^volumn ratio. 

"on a different Exsil" ODS column. 



94 



u 

o 

CO 

o 



a 
o 

o 

Pi 

■^ 
li 
<U 
&, 

o 
<o 

H 



u 

I 





_o 

^ 

c 
o 
O 

"c 


O 


ex 

E 

CD 
CO 



E 

CD 

c 


Z 




!c: 
^-< 

a 

c 
'i— 

T3 

"o 

CD 

O 

U-« 


o 

CD 
CO 

o 


a 

CD 

CD 

11 
< 



to 
to 

JC 

a. 


5 

to 
to 
> 

CD 

c 

CD 




phase A changed to 50:50 water:acetonitrile by 
addino acetonitrile 


o 
in 

6 
in 

g 

•o 
o 

CD 

o 

"S 
o 

CD 

d 
II 

< 


CO 
CD 

.c 
a. 


water:acetonitrile; phase B = 0.01 M ammonium 
acetate (pH5.72) + 0.1% acetonitrile in water 


phase A = 0.1% acetic acid in 50:50 

water:acetonitrile; phase B = 0.01 M ammonium 

acetate (pH5.72) + 0.1% acetonitrile in water 


o 
in 

d 
in 

c 

"O 

o 

CD 

o 

"5 

CJ 
CD 

d 

II 

< 


to 

to 

Q. 


wateracetonitrile; phase B = 0.01 M ammonium 
acetate (oH5.72) + 0.1% acetonitrile in water 


o 
in 

d 
in 

_c 

TJ 
O 
CD 
O 

"S 

O 
CD 

d 

II 

< 


to 
to 

SI 

a. 


water:acetonitrile; phase B = 0.01 M ammonium 
acetate (pH5.72) + 0.1% acetonitrile in water 


phase A = 0.1% acetic acid in 50:50 

wateracetonitrile; phase B = 0.01 M ammonium 

acetate (pH5.72) + 0.1% acetonitrile in water 












o 

CD 


o 


o 
o 


o 
00 


o 


o 
o 


o 

00 


o 


o 
o 


O 
00 


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o 
o 


o 
00 


o 


o 
o 


o 
00 


o 


o 
o 












O 


o 


o 
o 


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CO 


o 


o 
o 


o 

CD 


o 


o 
o 


o 

CO 


o 


o 
o 


o 

CO 


o 


o 
o 


o 

CD 


o 


o 
o 






o 
m 


o 
in 


O 

in 


o 
CO 


o 
o 


o 


o 
in 


o 
o 


o 


o 
in 


o 
o 


o 


o 
m 


o 
o 


o 


o 
m 


o 
o 


o 


o 
m 


o 
o 


o 






o 

CO 


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o 
o 


o 


o 
o 


o 


o 

CM 


o 
o 


o 


o 

CM 


o 
o 


o 


m 

CM 


o 
o 


o 


m 

CM 


o 
o 


o 


in 


o 
o 


o 






m 


o 


o 
o 


in 


o 


o 
o 


m 


o 


o 
o 


in 


o 


o 
o 


m 


o 


o 
o 


in 


o 


o 
o 


in 


o 


o 
o 






o 


o 
m 


o 
in 


o 


o 


o 
o 


o 


o 


o 
o 


o 


o 


o 
o 


o 


o 


o 
o 


o 


o 


o 
o 


o 


o 


o 
o 






c 

E 


E 


< 


CQ 
55 


E 


1 


< 




'e 


E 


< 


CQ 


E 



E 


< 

35 


CQ 
S5 


E, 


1 


5 


CQ 


"c" 

'e 



E 

•4-1 


< 


CQ 

55 


E 


1 


< 


CQ 
55 






< 


to 

CD 
Q. 

3 

o 

E 


CD 
CL 


a. 

E 

CD 
to 


CO 

o 

Q. 



Q. 

E 
to 

(0 


o 

Q. 



CL 

E 

CD 

to 


CQ 


to 
CD 

a. 

o 

E 


CO 

CD 
Q. 



Q. 

E 

CD 

to 


CM 

O 

CD 
Q. 

a. 

E 

CD 

to 










CD 

CN 

O) 

5 

LU 
CO 




( 


5 




( 

1 
( 


o 
<* 

5 

u 
/i 






■o 

Si 

V— 

a 

CO 




( 




i 

Xl 

/) 






CM 

u 






Si 
O 

LU 
CO 














■o 




X 




o 













a. 




3 




to 




E 


c 


3 


o 


to 




(O 


_) 


CO 


o 
to 


o 

Q. 


to 

3 


■a 


o 


o 





to 


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cr 


.c 


r 


5 




O) 


CD 


c 






r 


o 




(D 


"5 


2 


c 


>% 





n 


Q. 


CD 


O 


Q. 


F 





*-., 


n 


E 


E 
to 


T- 


to 


o 


F 


L. 


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0= 


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C7) 


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E 


b 



95 



Table 6-6: The products of recombination including dimerization after penicillin G 
reacting with potassium superoxide. 



OH 



CH3 s- 

/ 



-OH, 



~S CH, 



OH 

296=149+149 
dimer of Penicillamine 



H3C- 



H2N 


/ 



-CH, 



~S CH 



OH 

312=296+16 




^ — ( )— chj:h3 nh, 
0=^ SH3C 

OH ■' " ■ 

632=296+354(352+2H)-H20 
352=Benzylpenicilloic Acid 




642=308(Benzylpeniltoic Acid)+ 
334(Benzylpenicillenic Acid) 



Mechanism: 

RSH + B- 

RS- + O2 

RS- + 02- 

2RS 
BH + Oi^ 



RS- + BH 

RS + Oj- 
RS + 02^ 



-*- RSSR 



-.► OH- + B- + O2 



96 



Table 6-7: The major products of hydrolysis of penicillin G after interacting with 
potassium superoxide. 



Mobile Phue A 
[0.1%HOAcin 50 50H2OACN] 



MobilePhiseB 
P 01 M N»IOAc(pH 572) +01% ACN] 



Compound 



RT7.79 min 



RT19.94min 



benzylpenicilloic acid 

C.jHaoNAS, MW352 

[M+H]*=in/z 353.4 




RT9.58 min 



RT20.08 min 



benzylpenillic acid 

C,6H,8N204S, MW334 

[M+H]*=m/z 335.4 



HO — f^ 

\..-^ — / CH 



H, 
■CH, 



Qr <^ 



RT 10.09 min 



RT20.91 min 



ben2ylpenicillenic acid 

Ci6H,8N204S,MW334 

[M+Hr = m/z 335.4 




RTl 1.77 min 



RT22.15min 



penicillin G 
Ci6H,8N204S,MW334 ">"= 
[M+Hr=m/z335.4 




RT12.26min 



RT22.59 min 



benzylpenilloic acid 

C15H20N2O3S, MW308 

[M+H]+ = m/z 309.4 



/ •■^- 




97 



Table 6-8: The major products of oxidation of penicillin G after interacting with 
potassium superoxide. 



Mobile Phase A 
[0.1%HOAc in 50:50 H20:ACN] 


Mobile Phase B 
(O.OIM NH40Ac(pH5.72) + 0.1%ACN1 


Compound 


RT11.54min 


RT20.74 min 




benzylpenilloic acid sulfoxide 
C15H20N2O4S, MW324 
[M+H]*= nVz325.40 


RT11.57tnin 


RT20.56 min 


* 

.0 
HO^ 

Y^N~^ CH3 

benzylpenillic acid sulfoxide 
C,6H,J^20sS, MW350 
[M+H]* = m/z351.39 



98 



Table 6-9: The six p-lactam antibiotics that emit no chemiluminescence. 



O 



%ll 



HN- 



O 



/ 




HO V O^ 



\ 



o 




azetidinone 

MW71.08 

C3H5NO 



sulbactam 

MW233.24 

CgHuNOsS 



clavulanic acid 
MW185.18 
C8HnN04 



H,C 




HjC. 
H,C' 




,NH ^^^3 



^^ 




„^ 



V 



OH 




methicillin 

MW3 80.41 

CnH^oNiOsS 



Penicillin V 

MW350.39 

C.sH.sNjOsS 



"Y 




HN 



°/ 

OH HjC 

cefotaxime 

MW455.46 

CsHnNsOySz 




^ 



NH, 



99 



Table 6-10: The seven P-lactam antibiotics that emit chemiluminescences. 



Antibiotic 


Structure 


Relative Intensity 


Penicillin G 


°<)M 


100 


Dicloxacillin 


:?p^- 


54.4 


Piperacillin 


o 


47.9 


Anipicillin 




43.3 


Cephalothin 


"T^^ 


8.3 


Amoxicillin 


OH 


5.6 


Hetacillin 


OH 


0.6 



100 






TO 



U] 
o 












ux.. 



10 



20 



Time (s) 



Figure 6-1: The profile of chemiluminescences of penicillin G in different solutions with 
varied deuterium oxide to water ratios. 



101 




1= 

15 i 



o 

Q 

a 

5 



o 



+-» 

O 

o 



90ft 
SZC'I. 







8 



I 



102 



9e>'62T- 







103 







^-^ *4.-,. 



104 











a 
o 

X 
O 



s- 




i°o 3 

I • "^ 

1° ° 

I a M 

J " "5 

>- O 

° (-1 

a, 

1 2 C 

J ' .2 



105 




106 



^:^.^ 



OOS'T—. 



A^ 



►ES'S- 



TIT 



J 



HD 



o 

Q 

.a 
> 

(3 
1- " 3 



D 



li 






CO 
O 




1° 3 

1^ : 






tL, 



107 






Et9'99_/ 



lf::>^" 



108 



I6»-99^^ 




109 










r~^ 



6S8'»_7~ 





1; 






Pk 



no 




Ill 




57 mm 
43 mm 

After the third run ^'^'^"^ 
28 mm 



and spraying 



22 mm 



6 mm 



I 



covered 
by paper 



100 mg/ml of sample PGKO2 



solution 3(y) 



solution 2(y) 



solution l(p) 




After the first run 
and spraying 



47 mm 


. 


19 mm 


• 


12 mm 


• 


4mm 


• 







solution 3(b) 

solution 13(p) 
solution I2(p) 
solution ll(p) 



C: solution 1 
D: solution 2 
E: solution 3 



p: purple 
b: brown 



Figure 6-12: The schematic diagrams of TLC assays on sample PG and sample PGKO2 



112 




55 mm(y) 



The first run, and spraying 
(1) ninhydrin, (2) iodine-azide 21 mm(y) 



6 mm(p) 




The second run 
and spraying 







62 mm(y) 
58 mm(y) 








58 mm(y) 


■■"- 


''-"' 


53 mm(y) 
46 mm(y) 


' -' 'I' 


53 mm{y) 
46 mm(y) 




50 mm(y) 


-''-'■ ''-"-' 


40mra(p) 


» 






38 mm(y) 


■'. -' '' ' 




— 




The third run 




^ 


30 mm(p) 




and spraying 


23 mm(p) 


19nim(p) 


t 


- - 




16 mm(p) 


• 









solvent front 



44 mm(y) 



21 mm(y) 



58 mm(y) 
55 mm(y) 
50 mm(y) 

38 mm(y) 



A: 10 mg/ml of sample PGKO2 p: purple 
B: 10 mg/ml of sample PG y: yellow ■/ 



Figure 6-13: the schematic diagrams of preparing the samples for mass spectroscopy by 
the TLC separations. 



113 



a\ 






o 


vo m 






o 


rH rH 








rH ■»)l 






A 


o vo 






O O 


o w 






■«» ro 


(V 






in 0) 


to JJ 




JJ 


<s>X 


U M 




0) 


(0 n) 


nJ iiJ 




i-H 


01 (1) 


rH 4J 




C 


« a 


u cn 




H 


S* 


r» 








<n 








n 








3 








f 








r- 








o 


rr^ 




vo 
in in 

n rH 
<N rH 




X 


2 
M 






D 








,_ 




4J U 




u. 




C H 




X 




M PS 




u 


• • 






g 


4J 

s 

•H 






«o 














a: 




3 








S 




)j 




6 




UJ + 








S 






10 


M W 




o 


JJ 


cn > 




o 


c 


J + 




o o • 


IN 






■ • o 


O 


en m O in in o 


r^ m 


O < 0. 


o o o 


>H iH 


0- b- S 


rH rH rH 



ugg'OaiiaSHie 
woiusotnzo- 




114 



tueo — 



VI III -^ I r 



K !5 

'^ .1 IT' I 



' . I = ^ , ||1C| uf |g= 



CVJUJU 

w tk U. Iv* ^ ^ Co 






r 



M 




"T — r 



-Y^ 



93 



,1 .1.1,1 J "^1 I n |l|g 



T-nr 



Qc _ • 

<X <S S> (S Si 9 ® SI •-• 



4cr\isoja»CDC\j'^9 
r> rs oj r\ w rs o& 



« ■-• rj ro ** *^ 00 «^ o^ ^ 

.u. .VH-incotOftjmooojffs 

:i£ <roc 

— w cu M ro if> ■«• »- 



TT-r 



;si 






Figure 6-15: The chromatograms of sample PG (a) and sample PGKO2 (b). The buffer 
system: pH5.66 phosphate buffer containing 0.1% acetonitrile. 



115 



Reduction 



000, 



© 



® 






© 






Electrons 



Oxidation 



ii 




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Electrons 



Figure 6-16: Schematic of major processes occurring in electrospray. 



116 



ui „ 60606 

J*" *:> n fsi «i ■**' -^ 






c 

3 

CO 



o 



"8 



H 



s 







o> 



CT_L 

Wo. 

61 

Q,CO ( 

3o ' 

■S«)' 

Oro 

S > 
Ug 




g"'^"'^'"S" 



m 



a 



aoucpunqy ui|*|stj 



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or-t 

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P 

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Bo 

Is 

^1 



uj ^60606 

•i^ NlrilOOCJh^ 
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KMiepunqy win'ftl 






117 



CM 



CO 

<o 



..... <r> . + '? 
,00000 LU,, tocoio 

.▼- bcococococo 2c» ccococo 



in 



II 



ui 

CM 

2 






<p 




CO 


NL: 
3.81 E5 

334.6- 
335.6+ 
351.6-352 


to 

UJ 


IS 


Zh-" 





CO 




o> 




O 


u> 


•? 


III 


II «? 


..■<»■ 




O 






rcM 






CM 










i 


CO 




o 


CM 






CO 




S 


—■^ 




Cy 


CM 




CO 






J3 


■* 




iJ 


CM 




cct 








^ 




CO 


CM 




^-* 






§ 






-§ 


p 




^ 






CX 


CM 




o 






r3 


CO 


E, 


•I— 1 

X 




ID 


o 






i 


CO 




rt 


^ 




1 



aouepunqv sAjiBiay 



118 






Compound 350b 
MW350 



Oxidation 



Benzylpenicillenic Acid 

MW334 

RT20.91 mia 




Benzylpenicilloic Acid 

MW352 

RT 19.92 mia 



Oxidation 



Compound 368 
MW368 




Oxidation 



Penicillin G 

MW334 
RT22.15min. 







BenzylpeniUic Acid 
MW334 
RT20.08 






Benzylpenilloic Acid 

MW308 

RT22.59 mia 



Oxidation 



P.. 

o 

Compound 324 
MW324 
RT20.81 




Compound 350c 
MW350 



Oxidation 



Compound 350a 

MW350 

RT20.08 mia 



Compound 350b, 350c, 368 are detected by different HPLC gradient. 



Figure 6-19: The scheme of generation of sulfoxides after penicillin G reacts with 
potassium superoxide. 



119 




r 
z 

< 





:72 4> 



pa 



^i*" 









z 
I 


O '^ -~- 




^s^ 


'^o 


o ^ 



. ^o 



^ 




o 






■^ 




* ^° 




vk 



1/1 

O 

>^ 

x> 

o 

■?< 
o 

o 



o 



o 

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ex 
o 

a 



o 



o 
l' I 



CHAPTER 7 
SUMMARY 



The conversion mechanism of superoxide anion in aqueous system 
can be described as the folio wings: 

Superoxide dismutation: SOi' + 2H* »- H2O2 + OiCAg) 

Haber- Weiss Reactions: H2O2 + 02" — ^ OzCAg) + OH" + OH 

H2O2 + OH .► H2O + H02- 

H02- ^ H" + 02- 

OH + O2- ^ 02('4) + OH 

Quenching reaction: 02('Ag) + 02" ^ ^Oj + Oj" + 22 Iccal 

Chemiluminescent reaction: 02('Ag) *- ^02 + hY(1270nm) 

U + H2O ^ 2H2O2 

The superoxide anion has been proved to be relatively unreactive toward most 
biological components. This is supported by the observations that no chemiluminescence 



120 



121 



was detected when superoxide anion in 18-crown-6-ether-acetonitrile was used to oxidize 
penicillin G in acetonitrile, methanol, butanol, and chloroform. In these solvents 
superoxide anion will not readily convert into other reactive forms and these observations 
indicate that superoxide anion itself is not capable of oxidizing penicillin G with the 
emission of chemiluminescence. However, in aqueous solution, through the superoxide 
dismutation reaction, superoxide can generate reactive species, for example, hydrogen 
peroxide and singlet oxygen ('O2). Hydrogen peroxide is used extensively as a source of 
^Oj and it is not reactive enough to oxidize the thirteen P-lactam antibiotics with the 
resultant emissions of chemiluminescence. This leaves 'O2 as the most probably reactant 
for the oxidation of penicillin G and the emission of chemiluminescence. The 
involvement of 'O2 in the chemiluminescent reaction of penicillin G after reacting with 
potassium superoxide was strongly indicated by the D2O experiment using deuterium 
oxide instead of water as the solvent for penicillin G. Meanwhile, no chemiluminescence 
was measiu-ed with penicillin G in phosphate buffer. It is believed that 'Oj is probably 
quenched by phosphate buffers. The experiments had indicated that pH4.5-7 is the best 
pH range for the chemiluminescences of p-lactam antibiotics because they are stable in 
the solutions with pH values close to neutral and the superoxide is easily converted into 
'O2 by superoxide dismutation reaction. The degradation studies of penicillin G by 
HPLC/ESI(or APCI)-MS/MS suggested that the chemiluminescence of |3-lactam 
antibiotics was generated by the autoxidation initiated by 'Oj. This type of reactions is 



122 



also the pattern of luciferins and many related compounds. The maximum wavelength at 
535 nm of chemiluminescent penicillin G is very close to that of Lucifer's at 562 nm. The 
low efficiency of penicillin G's chemiluminescence is consistent with its chemical 
structure and the fact that superoxide is a highly efficient 'Oj quencher. The presence of 
'O2 was also proved by the characteristic 1268 nm chemiluminescent emission spectrum 
of 'O2 as discussed in chapter 1 . On the contrary, no detectable hydroxyl radical was 
involved in the degradation of penicillin G, because no aromatic hydroxyl substituted 
derivatives was observed by NMR and mass spectrometry. Perhaps, the hydroxyl radical 
can be postulated to be a short-lived transient intermediate, which is produced by the 
Haber- Weiss reaction, however, the data collected in this dissertation reinforce that 'Oj 
rather than hydroxyl radical is the prime oxidant in the reaction system. Even though 
transition metals are available in vivo, the metal-catalyzed Haber- Weiss reaction will face 
competition from Fenton-type reactions, in which H2O2 interacts with transition metals or 
metal complexes to generate hydroxyl radicals. Indeed, The questions arise which is the 
crucial toxic species, singlet oxygen or the hydroxyl radical, and whether the hydroxyl 
radical comes from the metal catalyzed Haber- Weiss reaction or from a Fenton-type 
reaction. The answer to these questions must be specified in order to better understand 
oxygen toxicity and develop new therapeutic strategies as well as new antioxidants. 






»-*.?■. 
W 



GLOSSARY 

APCI Atmospheric pressure chemical ionization 

APT Attached proton test 

ATP Adenosine triphosphate 

BHT Butylated hydroxytoluene 

CFFAB Continuous-flow fast atom bombardment ionization 

CI Chemical ionization 

CNS Central nervous system 

DEPT Distortionless enhancement by polarization transfer 

DMSO Dimethyl sulfoxide 

DNA Deoxyribonucleic acid 

EI Electron impact 

ESI Electospray ionization - 

ET Electron transfer (or exited state) 

FAB Fast atom bombardment 

FWHM Full width at half maximum 

GC Gas chromatography 

HPLC High pressure (or performance) liquid chromatography 

HV High voltage 

MS Mass spectrometry 

NMR Nuclear magnetic resonance 

ODS Octadecylsilane 

PB Particle beam 

PDA Photodiode array 

PMT Photomultiplier tube 

RSP Relative standard deviation 

SOD Superoxide dismutation 

TLC Thin-layer chromatography 

TME Tetramethylene 

TSP 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (or thermospray 

ionization) 

UV Ultraviolet 



123 



APPENDIX A 
THE PRODUCTS OF HYDROLYSIS OF PENICILLIN G FOLLOWING THE 
INTERACTION WITH POTASSIUM SUPEROXIDE - THE CHROMATOGRAMS 
AND MASS SPECTRA OF HPLC/ESI-MS/MS. 



, ..J' 



o 

-<M 

n 



O 



-o^ 



^ 






cf^ 



-1^ 



e^;t2 



0— p= 



l: 



to 

5 

to 
55 

0>CO 

"5-yjf 

■on. 

to to f 
So 
5 ml 

o™ 



SI 



!Kh 



1 1 Tllllllllllllll 
00 to rt C4 

83uepunqv 8Ai;e|au 




1 




°i 



aoii_ 



Kh 



1 T T-n I 1 I I [ I I I I I I 1 

o o o o o 

CO <D ^ OJ 

douepunqv OAiieis^ 




— T 1 T— — I 1 1 f- 

e S 8 S S S 2 



'B 
a, 



o 



00 



00 



> 

o 

CI, 
u 






125 



126 



308 - RT12.26 min - HPLC / ( + ) ESI - MS / MS 



(XX,: 



'-YX- 



C,4H„N20S 

MW262.37 

[M+H]* = 263.38 



^O^-^h; 



C8H9NO 

MW135.16 

[M+H]*= 136.17 



k.-^'^^./^.H 



rx- 




CsHjoNjOsS 

MW308.39 

\M+HY = 309.40 



C4H7NS 

MW101.17 

[M+H]*= 102.17 



*-\ 



C5H5NOS 

MW127.16 
[M+H]*= 128.17 



C,5H„N202S 

MW290.38 
[M+H]* = 291.39 



HjN* 




.CH, 



CHmNzOzS 

MWl 90.26 

[M+H]*= 191.27 



HjCi 



^^ 



CtHiiNOzS 

MW173.27 

[M+H]* = 174.28 



CjHiiNOS 

MW145.22 

[M+H]*= 146.23 



Figure A-2: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic acid. 



127 



o 



o 



m 

6. 




O 

C3 

O 
O 



^-' ns 



ex 



It-I 
o 



CO 



00 



> 

u 

d 
u 

H 
t 

< 

i 

•I-H 

fa 



128 



308 - RT12.22 min - HPLC / ( - ) ESI - MS / MS 



(^ 




H, 



C15H20N2O3S ho' 
MW308.39 
[M-H]- = 307.39 







H, 



C,4H,8N20S 

MW262.37 
[M-H]- = 261.36 



^ 




CuHhNzOS 

MW234.32 

[M-H]- = 233.31 




CnHioNjOS 

MW230.28 

[M-H]- = 229.28 



Figure A-4: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilloic 
acid. 



129 



;:\LCQ\data\Spec\seq1924f 09/12/97 04 0304 

m phase C18 HPLC/ESI-MS 

>#: 809-810 RT.iiM-iiM A\/: i ^5 6: S i2 00-i2.62 22 34-22 39 NL 

r: ♦ c ms ( 75.00 - 1200.00] 



Penicillin G [8/28/97. Sun] 




352'f-CM + NH+]* 



il'vl;-' 



685 



3W + 335 

587 <-! 
^1 



K-\ 45g.476i96 569 64,2 g^9|| ,_IP6 ^^^ 335 917 995 1026 1Q61 1117 1140 

■»00 500 6(1)0 ' ' ' ' 7(1)0 ' ■ ' 'edio' 900 1000 ' 'n'oo' ','■>', 



iT. 19.35-24.70 
100 



1100 



1200 



22.13 



8 0^ 
S100-, 

C 

I 50^ 

> 

a 
Sioo^ 

50-^ 



NL- 

1.16e7 

BaMPeak 

20 06 / \ '"'^' 

19.62 ^^^^ 20,5 20^ .00 ,,,, ,,,, / V ,,^ ^^^ ^^^^ ^3^ ^3^^ ^^^ ^^^^^ ^^^^^ ^^^ ^ -— 

"lis ^^ ^ NL: 

2.02E7 
mfta 

334.S-335.S+ 
20 06 / \ 351.5-352.5 

19.62 19,89 ^ 20.35 20.55 20,93 2^,07 21.56 21.81 / V^ 2Z43 22.68 23^ 23.42 23.55 24.10 24.32 24 59 

22?I5 NL 

1.81 E6 

m/2« 

308.6-309.6-* 




19.92 



20.87 



19.76 .' \ jO^OS 20 35 



.-^ 



4i:??.ii35 21,65 2K93 



100 





22.59 



22.11" 



V22.79 23,13 J^^Jk^ 



,2jj6 24.12 24.50 



19.5 



20.0 



20.5 



20.39 
21.0 



I 22,24 



^MLJMl^W 20,48 20.73X21.17 21.56 ''5ili^!^ " ,22.84 23.17 2^33 23. 70 24.OI 24.41 



r. 



2<.5 



22.0 
Time (min) 



22.5 



23.0 



23.5 



352.6-353.6 



NL: 

8.43E5 

m/z= 

350.6-351.6* 

367.6-368.6 



24.5 



C:M.CQ\data\Spec\seq 1 924v 
rev phase C18 HPLC/ESI-MS/MS 
!>#:S31-537 Kl : 11.77-11.90 AV- ? HL- 3 lyg' 
T: ♦ c Full ms2 335.10 [ 90.00 - 400.00] 

160 



100- 




^ 80-; 
















c 60- 




§ 




40- 












■ 




a. 20- 




- 


114 


qJ 


. 



100 120 



15a 



176 



140 160 



180 



09/13/97 02:12:26 



PenicJIIIn G 



307 335 



2<i0 • 2i0- ■ '2^0'' 2d0- ■ '2^0 '3io' ' ' 3^0 ' ' ' 3I0 '^ ' 3^0 ' ' '3^0 



40O 



Figure A-5: The positive HPLC/ESI-MS/MS of penicillin G and MS/MS of m/z 335. 



130 



\LCQ\dala\Spec\seq 1 924M 
V phase C18 HPLCyESI-MS 

*: 501 RT: 11. 8S AV: 1 SB: 7i 4.53-5.05 NL 9.2dE5 
- c Full m$ [ 75.00 - 800.00] 

lOOj 

i 60^ 

I 50^ 119 

p 

10^ 



09/12/97 18:58:26 



Penicillin G 



83 



m 



157 192 



289 



331 



667 



^M9 



J50 390 



4p9 



3^«^1A15 458 



639 



2'<333tl 



558 

683 



<6o Uio ' 560' '5io' ' ' 'eio' ' ' 6io' '71 



701 
'700' 



P7 742 258 

' , ■■■■■ , [ , ', . 



100 



250 



360 



350 



m/z 



7io ' sio 



T: O.O1 - 30.03 



100 
50^ 

I " 
|100t 

» 

I " 

- lOOn 

50^ 



100 

50- 

o-l— . 



11.89 
0.00 



0.23 

00 '-'^ 
^0,00 



2.92 3.70 4.91 
0.00 0.00 0.00 



6.52 7.14 
.00 fl.00 



O.M^ 



^n°;i^ "i^ro^fi? ";" 1i£? 16.31 17.41 



.00 n 



.00 ojDO ^00 oQQ 0,00 00 



20.47 21.24 22.57 24.51 
0.00 



27J1 28.43 
O.Qp OjO_ 



11.89 
333.44 



10.22 
333.03 



12.15 
333.04 



10.07 
307.04 



12.39 
307.06 
13.22 
307.02 



11.91 
351.16 



jS!L.hiii 



10 



10.98 
351.00 n 



12 



-»•■■■•■ .r 



14 16 

Trme (min) 



" I ' 
18 



1 ' • 
20 



•r I 

22 



IT 



^ 



IT 



NU 

5.31 E« 
BasePeali 
miz' 
7$.M00.0 



NU 
a.30ES 

333.0-334.0 



2.68E4 

m/z» 

307.M08.0 



NL; 
4.12E4 

351.0-352.0 



30 



:::\LCQ\dala\Spec\seq 1 924w 

ev phase C18 H PLC/ESI-MSA^S 

5* 527-534 m 1169-11.84 AV: 8 NL- 9 73E4 
r: - c Full ms2 333. 10 ( 90.00 - 400.00J 

100 



8 80- 

c "• 

m 

I 60 

m 

K 20- 



120 



09/13/97 18:45:54 



192 



180 



2<lo' ' '2io' 



2^0 ■ '2^0' 
m/z 



Penicillin G 



289 



288 

2^0 'sio' 



320 



ur 



3io '3^0' 



Figure A-6: The negative of HPLC/ESI-MS/MS of penicillin G and MS/MS of m/z 333. 



131 



Penicillin G - RT22.15min - HPLC / ( + ) ESI - MS/MS 




C,6H„NAS ' 
MW334.39 
[M+H]* = 335.40 




^^=0 



C,oH,N02 

MW175.18 

[M+H]^= 176.19 




CsHsNOzS 

MW159.20 

[M+Hr= 160.21 



HjC 



CHjNS 
nVzl 14.18 



Penicillin G - RTl 1.89 min - HPLC / ( - ) ESI - MS/MS 



H0~^ 




CsHlNAS 

MW334.39 

[M-H]- = 333.38 



CjHtO 
m/zl 19.14 



■s><x(. 



C,5H„N202S 

MW290.36 
[M-H]- = 289.35 



^ Ovh- 



C„H,5N02 

MW193.25 

[M-H]= 192.24 



Figure A-7: The interpretations of the positive and negative HPLC/ESI-MS/MS of 
penicillin G. * ^ 



ri.. 



132 




8f 



TTTT 

o 



1 1 1 1 1 1 1 1 1 1 1 1 r [ 1 1 ) 1 1 1 [ p 1 1 m 1 1 [ 1 1 
o o o o o o o 

CO h- (O U> Tt fO CVI 

30UBpunqv aAjjeiay 



CO 







1 " " I " " I " "I "" 1 < " 1 I M I I I I 

S 9 2 o Q o o 
o OT CD r~ <o tn ■« 



aDuepunqy 3Ar)e|a^ 









o 

I 

I— ( 
CO 

O 



> 



CO 

O 

D, 
(U 

S3 
H 

do 

< 

I 



133 



334a - RT20.33 min - HPLC / ( + ) ESI - MS / MS 



r 

CsHi^NzOjS 

MW3 16.37 

[M+Hr = 317.38 






C16H18N2O4S 

MW334.39 
[M+H]* = 335.40 



o 
C,HuN04S 
MW2 17.24 
[M+Hr = 218.25 



.0 T 

,CH, 

.. ■ "^ 

HN*= 

CyHnNOaS 

MW173.23 

[M+H]*= 174.24 








C6H9NO2S 

MW159.20 

[M+Hr= 160.21 



.0 




.CH, 
CH, 



r^ 



CisHijNaOzS 

MW290.38 

[M+Hr = 291.39 



C5H,NS 

MW115.19 

[M+Hr= 116.20 



Figure A-9: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenillic acid. 



,f: 



134 



E 
o 



O 

a. 






o 
o 



o 



CO 






0)f 



5^ 

LU 

CO o^ 



WI 
raZ 

Is 

o 2 



CM 



UJ 
Q 



Sl 



-L- 



Sf" 



- $2f 



Mnip 



I < I " ■ 1 1 " " I " " I " " I " 
o o o o o 

03 r- <D IT) -^ 






o 

-CM 
CM 



> — o 



iuOLEiioo 

i^'^ J= =><00<M 



10 






o 

1$ 



CM\. 
CM 

u 

81- 




o o o o 

CO CM •>- 



k/)u. 



oouepunqv aAijeiay 



i""i""i""i""A""A""A""i""i' 

0<7)<Ot^u5i5-«(OCM 



o 

CSJ 



o 

ID 



E -? 



93uepunqv wiiieiaij 



o 

C/3 



C/D 



00 
W 

u 



> 

• i-H 

CO 

O 
D. 

(U 






135 



334b - RT21.51 min - HPLC / ( + ) ESI - MS / MS 



HS — \ — ^^ 




C,6H„N204S 

MW334.39 
[M+H]* = 335.40 



CsH.sN^O^S 

MW288.36 

[M+H]* = 289.37 



Q\ 



CHioNzOj 

MW202.21 

[M+Hr = 203.22 



HS — 1 — '-"s 



HjC; 



-Y 



CsH.oNOzS 
m/zl60.21 




\ / 



■oAo 



C,Ji,N02 

MW175.19 

[M+Hr= 176.19 



HO 

H,C NH* 

^ // 

MW127.14 
[M+Hr= 128.15 



CsHi.NO 

MW113.16 

[M+Hr= 114.17 



Figure A-1 1 : The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicillenic 
acid. 



136 



-s 



o 

-eg 



gf 



ino 

..o 



1^ 



cnco , 

■&iy ! 

Q)0 ( 

i2-J 

oOl 

Bo 

■§'®F 

8ft 






I Ml 1 I I I 1 r I 1 r-T-pi 

o o o o 

00 to -Tt Csl 

aouepunqv aAijeiay 




souepunqv 3Ai)e|3H 



i?H 






15 



2<^ 



^^ 



"^1 




3 

a 

«» ■ 

IB - 

oi ' 
o 
"« 



8, 









¥^T' 



o 

C3 






o 

c 

o 
00 



00 



U 

u 

_> 
*■♦-» 
a 

u 
H 






137 



334b - RT10.09 min - HPLC / ( - ) ESI - MS / MS 




C,6H,8N204S 

MW334.39 
[M-H]- = 333.38 



*> f . 



:"v«._ ?^"5 :. 



;* . '^ 



\'"'- ;"%<; 




C.sH.gNjOjS 

MW290.38 

[M-H]- = 289.37 




C,5H,6N202S 

MW256.30 
[M-H]- = 255.29 



Figure A-13: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenicillenic 
acid. 



138 




o 
o 

'B 

'o 

•a 

u 

ex 

u 
x> 

o 

00 



C/3 



00 

u 



aouepunqv aAiieja^ 



'■4-» 

O 

a 
H 






139 



352 - RT19.49 min - HPLC / ( + ) - ESI - MS / MS 



CH, 






CsHjoN^OjS 

MW308.39 

[M+H]* = 309.40 




C,3HhN202S 

MW262.33 
[M+Hr = 263.33 




CsHzoNzOjS 

MW352.40 

[M+H]*= 353.41 




CjH|,N04S 

MW2 17.24 
[M+Hr = 218.25 




C6H9NO2S 

MW159.20 

[M+Hr= 160.21 




C,6H,8N204S 

MW334.39 
[M+H]* = 335.40 






C,H„N02S 

MW173.23 

[M+Hr= 174.24 



n4 

( 
s 



CsHsNS 
m/zll4.18 



Figure A- 15: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicilloic 
acid. 



140 



o> 

B 



O 



o 

Q. 



iii 



a. 

X 

00 

O 

SI 

^ 

(DO 

(QI 

raZ 



ro 



Ora 
9^ 



C4 



H 



CM 



lO 



5° 



CDO 
O 



W 

E 



ym I imifiTi I inTrprT 
o O o o o 
o o) CO r^ <o 



lyrrrrTrrrrprnjTTnjnTi 
o o o o o 

lO Tj- CO oi ■•- 



o 

-<D N 

'1 



90uepunqv aA{)e|3^ 



QJ u_ E*-oo 




O 

o 

O 

•a 



— : • s 



33uepunqv 8At)e|3^ 



X> 

o 

00 



00 

I— I 
00 

u 
-1 



c 
H 



i 



141 



352 - RT18.36 min - HPLC / ( - ) ESI - MS / MS 




C,5H,5NO,S 

MW289.35 

[M-H]= 288.34 




A 



C13H12N02S 

MW245.29 
[M-H]- = 244.29 




CHijNAS 

MW260.31 

[M-H]- = 259.30 




MW352.40 
[M-H]- = 35 1.39 




C.sHjoNjOjS 

MW308.39 

[M-H]- = 307.39 




/ 

CsH^N^O^S 

MW170.18 

[M-H]- =169.18 



C,4H,J^203S 

MW292.35 
[M-H]- = 291.34 



r^ 



CsH.jNjOjS 

MW2 16.25 

[M-H]- = 215.25 




C,2H,4N20S 

MW234.23 
[M-H]- = 233.31 




C„H,oHO,S 

MW274.29 

[M-H]- = 273.28 



Figure A-17: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenicilloic 
acid. . ■• 



'.'> ** i> '> 



U * 



APPENDIX B 
THE PRODUCTS OF OXIDATION OF PENICILLIN G FOLLOWING THE 
INTERACTION WITH POTASSIUM SUPEROXIDE - THE CHROMATOGRAMS 
AND MASS SPECTRA OF HPLC/ESI-MS/MS. 



i: T ^ 



vr;,--^ 



o 



C3 



^' 



CO 



! a> 
: to 






CI 



O) 



0) 



So 

CO o 

So 

tx-<r 
cox 

roZ 

li 
8f 



§/ 



CD<N 



1 1 1 1 1 M 1 1 1 1 1 1 n 1 1 



O 2 



It .. 



Hjl IIM I II || II II I II I 1)1 I 

~ o o 

CO CM 



to lO 



TJTT7T 



r<^ 




■f 


+ <N — 




"O .. "o • O 




lu u, Eooo 




J^ObW^o 


o 


2 V- 1— U-rjoO"* 


-o 




.^ 





souepunqv 9Aj)e|a^ 




r^ © m 



&, 

o 
u 

X 

<B 

3 
(/I 

u 

■♦-» 

tt-l 
o 

t/3 



00 



C/3 

U 

H-l 



> 



o 
cx 






eouepunqv SAiieis^j 



IX, 



143 



144 



312 - RT28.16 min - HPLC - ( + ) ESI - MS / MS 



HsCv^^H^ 



HjN* 



OH 



C5H7NO2 

MW113.il 

[M+Hr= 114.12 



H3C 



C4H„NOS2 

MWl 53.26 

[M+H]*= 154.26 



H,C 



CoH.sNjO^Si 

MW294.38 

[M+H]* = 295.39 





C5H6NOS2 
ni/zl60.26 



OH 



Hjn: 
CH3 \ 

/ 

-S 



-CH, 



CH, 



H^ir-^^ 



CoHjoNjOsSa 

MW3 12.40 

[M+Hr = 313.40 





C^HgNzOzSz 

MW204.26 

[M+H]* = 205.27 



'SH 



H2N* 



C2H5NS2 

MWl 07. 19 

[M+Hr= 108.20 



H3N* 



OH 



H,C- 



O 

CH3 \ 
/ 
-S 



OH 

QH,6N205S2 

MW284.34 
[M+H]" = 285.35 



Figure B-2: The interpretation of the positive HPLC/ESI-MS/MS of the sulfoxide of 
penicillamine dimer. 

...?.•/'■ •''..' ,\.r. 



145 




o 




rt^ 




■* 






•* .. i9o • o 








j«"3;^Sa 


o 


Zm 1-u.nco-* 



souepunqv aA{)e|3y 



^CV. 







aouepunqv 9A!}et3^ 



U 
O 

u 



O 
00 



00 



00 

u 

PL, 

> 



<2 



•t-4 

{X4 



146 



312-RT28.il min-HPLC/(-)ESI-MS/MS 



CH3\ 




H3C- 



HjN 



-CH, 



CH, 



OH 

CoHioNzOsSz 

MW3 12.40 

[M-H]-=311.39 



N 



./=\. 



% ^' 



CH3 

C6H5NOS2 

MW171.23 

[M-H]-= 170.22 



H3C- 



HjN 
CH3\- 

/ 

-s 




-CH, 



CH, 



^NH, 



C9H20N2O3S2 

MW268.39 

[M-H]- = 267.38 



Figure B-4: The interpretation of the negative HPLC/ESI-MS/MS of the sulfoxide of 
penicillamine dimer. 






147 




aouepunqv 8Ai)e|9^ 









o 

"a 

O 
C/3 



I 

pq 
U 

> 



CO 

O 
O. 
(L> 

fS 

I 

CQ 

(3JQ 



148 



324 RT21.83 min - HPLC / ( + ) ESI - MS / MS 




CHlNjOsS 

MW306.38 

[M+H]* = 307.39 



C,4H„N202S 

MW278.37 
[M+H]* = 279.38 




ChHzzN^O 

MW234.34 

[M+H]* =235.35 






CsHiiNOjS 

MW177.22 

[M+Hf =178.22 




C,5H2oN204S 

MW324.39 
[M+H]" = 325.40 



"!<«, 



K 



NHJ 



CHnNzOzS 

MW 188.24 

[M+Hr= 189.25 




C9H9NO 

MW147.18 

[M+Hr= 148.18 






CsHioNaOS 

MWl 58.22 

[M+Hr= 159.23 



"^ 



C4H6N2OS 

MW130.16 

[M+Hr= 131.17 



Figure B-6: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenilloic acid 
sulfoxide. 



149 




•i-H 

X 



O 

'o 

1 

a, 

o 
o 

CO 



00 

I 

00 

W 

U 

h-1 



> 

u 

d 
u 

XI 

H 

m 



aouepunqv dAiieia^ 



150 



324 - RT21.80 min - HPLC / ( - ) ESI - MS / MS 




C15H20N2O4S 

MW324.39 

[M-H]-= 323.38 



°\ CH, 



"w^^"' 



HH-^ 



C,4H2oN202S 

MW280.38 
[M-H]- = 279.38 




C,3Hi4N202S 

MW262.33 
[M-H]- =261.32 




C5H5NO3S 

MW159.16 

[M-H]-= 158.15 




CsH.sNiO, 

MW274.32 

[M-H]- =273.31 




C14H22N2O 

MW234.34 

[M-H]- = 233.33 



O N=CH, 

M 

O- CH, 



C4H5NO2 

MW99.09 
[M-H]- = 98.08 




C,4H„N20 

MW230.31 

[M-H]- = 229.30 



Figure B-8: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilloic acid 
sulfoxide. 



151 



9 



CM 
O 








to 








n 






«o 






- 


n 






CO 






h- 








SL 






▼* 




O 




° 




fO 














h- c3L 


- 




aouepunqv a/uieiay 





=?feo 




loSca 






CO 3O 


y- 


„u.° 


i^ 


^1^2. 



^ 



I5J 



■cHj'' 



q CD ,-- 
^ 00 ^^-=- 



■» l"iM"ii 




SDuepunqv 3A|)e|3)j 



152 



350a - RT22.35 min - HPLC / ( + ) ESI - MS / MS 



^{ 




C,6H,6N204S 

MW332.37 
[M+H]* = 333.38 




C.iHioNzOa 

MW202.21 

[M+H]" = 203.22 



"~0 

C^HhNjOsS 

MW322.33 

[M+H]* = 323.34 



CsHuNjCS 

MW350.39 

[M+Hr = 351.39 



CiHnNzOsS 
m/z305.37 



CHioNzOj 

MW260.33 

[M+Hr = 261.34 




^ 



CHijNOjS 

MW235.25 

[M+H]^ = 236.26 



C4H5NOS 

MW115.15 

[M+H]+= 116.16 




C,6H,7N204 

m/z301.32 









1 






r 




^ 


^ir- 




0>X" 


C6H9NO3S 






MW175.20 




C,5H,5N204 


[M+H]* 


= 176.21 




ni/z287.29 


V 


"V— «H. 


'\ 1 




J 


ivr-i 


C6H,N02S 


CH^NOjS 




MWl 59.20 


MW147.19 




[M+Hr= 160.21 


[M+Hr= 148.20 





Figure B-10 
sulfoxide 



': The interpretation of the positive HPLC/ESI-MS/MS of benzylpenillic acid 






153 



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OL 



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lU 



OT 


i:!'0 


rn <» 


-> 


u« 


UI • 


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cn = 

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t/)u. 



o 



in 

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SO 

CM 



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u I ri [ I I 1 I I I [ I r I I I I I I I I I I I I I I I 1 1 I I n I [ I I 

oooooooo 

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aouepunqv 3Ane|32j 



Jo O = oj9d 




^ 
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^ O 
N O 



1 ' ' ' ' I " ' ' M ' • ' I I I I ' [ I I I 1 J 1 I I T I IT 

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93uepunqv OAiieia^ 



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•i-H 




X 




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o 


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rt 




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a 


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sa 


H 




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CNJ 


(U 



IX, 



154 



350a - RT22.38 min - HPLC / ( - ) ESI - MS / MS 




\ 



H3 



CHnNOjS 

MW191.24 

[M-H]-= 190.24 




C,6H,6N204S 

MW332.37 

[M-H]- = 331.36 







C,5H,6N202S 

MW288.36 
[M-H]- = 287.35 




CH, 



C^H.gN^OsS 

MW350.39 

[M+Hr = 349.38 



C11H15NOS 

MW209.31 

[M-H]- = 208.30 



C,5H,8N203S 

MW306.38 
[M-H]- = 305.37 



CH.sNOjS 

MW241.30 

[M-H]- = 240.30 



CK^" 



CH, 



C,5H,5N,02 

MW256.30 
[M-H]- = 255.29 



CoHisNOS 

MW197.29 

[M-H]- = 196.29 



Figure B-12: The interpretation of the negative HPLC/ESI-MS/MS of benzylpenilUc acid 
sulfoxide. 



i . 



155 



E 



O 
Y. 



+ 
CD 



/S 



1 



Sl 



oo 

CM 

CO 



o 

<dO 

Q.Tt 

WI 
152 

Is 
81 



1_ 







Ko 
eg 



COlO 

o a> 

CMO 



2E 



CM D rrrrrnr 



^. 



9 






o o o 



90uepunqv aAjieiay 



.ciy§ .Dci^cg 



liO 
Z 1- 



(030 
o 1111.0 



Si 8- ' 



- Hir° 



L1_0 
OirtO 

- iiir° 



^ 



^ 




1^ (-» in 



■- 2 



£' 





X 



o 

a 

o 







o 



to 


(U 




a 






£ 


>» 


<D 

E 


a 

(t) 


fi"- 


^ 




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CZ) 




^ 


CM 


C/5 




S 




HH 




V) 




w 








u 




H-1 






W 


> 
















CO 









0, 


S 






H 



93uepunqv 8A|)e|3^ 



CO 

T— ( 

CQ 



156 



350b - RT24.13 min - HPLC / ( + ) ESI - MS / MS 



CsHisNiOiS 

MW332.37 

[M+H]* = 333.38 



O^ 



Ci,H,6Nj04 

MW232.2g 

[M+H]* = 233.29 



M_/y^^ 



C„H,oN202 

MW202.21 
[M+H]* = 203.22 



Cl4HMN20iS 

MW322.33 
[M+H]* = 323 34 



v. 

C,5H,6N20,S 

MW304.36 
[M+H]* = 305.37 




C,6H,^20iS 

MW350.39 

[M+H]* = 351.39 



HO 

HjC nh; 

CsHjNOj 

MW115.13 

[M+H]* =116.14 



CHijNOsS 

MW235.25 

[M+H]* = 236.26 



3 



»-""i 



•n 




CeHsNOiS 

MW175.20 

[M+H]* =176.21 




rT" 



CsHaNjO. 

MW260.25 

[M+H]* = 261.26 




«\ 



CsHsNOjS 

MW14719 

[M+H]* = 148.20 



CsHjNOjS 

MW159.16 

[M+H]* =160.17 



C5H7O2S 
m/zl31.17 




CsHisNiO. 

MW300.31 

[M+H]*= 301.32 



C,5H,4NA 

MW286.28 

[M+H]* =287.29 



Figure B-14: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicillenic 
acid sulfoxide. 



.' 'i . 




157 












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o 








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CO 


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- 


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V3 


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CM 




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CO 




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wo 


5° 




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:' 




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CM 




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fn 




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1 -J 1 1 1 M 1 1 1 1 1 1 1 1 M ; t ' : 1 1 1 1 ( ] ni 1 1 ri 1 1 1 1 1 
ITll. oooooooo 


1 1 1 1 1 1 1 1 M 1 r 


o o o 


^ ( 1 n Fi 1 M 1 1 1 n 1 ! ' ' " 1 ' 1 ' ' 


1 M 1 1 M M 1 1 1 i i 1 1 


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£ 


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it .'. aouepunqv sAijeisy 




^ aouepunqv 9/uiB|9a 












c; 













158 



350b - RT24.24 min - HPLC / ( - ) ESI - MS / MS 




s — O' 



O^H, 



H,C 



C,5H„N:03S 

MW306.38 

[M-H]- = 305.37 




C,4H,2N20jS 

MW288.32 
[M-H]- = 287.31 







C,6H,6N204S 

MW332.37 
[M-H]- = 331.36 



OH HN 



O- CHj 
S- 



HN 



C7H,3N03S 

MW191.24 
[M-H]-= 190.24 




\ // 






CH.gNjOsS 

MW350.39 

[M-H]- = 349.38 



HX 



%-V"' 



C8H7N03S 

MW197.21 
[M-H]- = 196.20 




C,4H,oN203 

MW254.24 
[M-H]- = 253.24 




OH 



C6H„N05S 

MW209.21 

[M-H]- = 208.21 



Figure B-16: The interpretation of negative HPLC/ESI-MS/MS of benzylpenicillenic acid 
sulfoxide. 



159 




a 

CO 

o 

Pi 



•a 

O, 
Cm 

o 

00 



00 

I 

00 

u 



douepunqv SAiieid^ 



*■*-» 

•i-H 

w 
O 

u 

H 



oq 



160 



350c - RT35.74 min - HPLC / ( + ) ESI - MS / MS 




C,4H,4N205S 

MW322.33 
[M+H]* = 323.34 



n HN--\ 

" 6 



C,5HnN203S 

iii/z305.34 




"^^: 




CisHnNiOj 

MW268.27 

[M+H]^ = 269.28 



C,4H,6N202 

MW244.29 
[M+H]* = 245.30 




C8H,2N204S 

MW232.25 
[M+Hr = 233.26 



C,6H„N205S 

MW350.39 
[M+Hr = 351.39 



C4H5O2S 
m/zl 17.14 



- :::>a/ 

t 

o 

C8H9N04S 

MW2 15.22 
[M+Hr = 216.23 




CgH,NO 

MW135.16 

[M+Hr= 136.17 




CsHtNOsS 

MW197.21 

[M+H]"= 198.21 



Figure B-18: The interpretation of the positive HPLC/ESI-MS/MS of peniciUin G 
sulfoxide. 



■Q -.^ 



:x'-^ 



161 




aouepunqv SAjieiay 



Z(0 h-U-COO)-* 




« 




'2 

CO 

o 

1=1 



'S 
o 

I 

t— I 
00 

O 



E 



> 

M 
U 



OS 

I 
PQ 

9J 



tM if 



ODUepunqv 8Ar)e|3u 



IX, 



162 



350c - RT35.95 min - HPLC / ( - ) ESI - MS / MS 




C,6H2oN204 

MW304.34 
[M-H]- = 303.34 




C,5H,8N204 

MW290.32 
[M-H]- = 289.31 




C,4H,4N204 

MW274.27 
[M-H]- = 273.27 




C.sHigNzOjS 

MW350.39 

[M-H]- = 349.38 




CsH.eNjOjS 

MW336.36 

[M-H]- = 335.35 




C|5H,4N204S 

MW3 18.34 
[M-H]- = 317.34 




C,3H,5N03S 

MW265.33 
[M-H]- = 264.32 




C8H,2N204S 

MW232.25 
[M-H]- = 23 1.24 



o- 



QHiiNOzS 

MW173.23 

[M-H]- = 172.22 



Figure B-20: The interpretation of the negative HPLC/ESI-MS/MS of penicilUn G 
sulfoxide. 



-., f ■ 



163 




O 






o 

t 

o 
o 

o 
00 



00 

I 

GO 

u 



aouepunqv SAne^a^ 



> 

•l-H 

w 
O 



I 

00 



164 



366a - RT21.62 min / HPLC - ( + ) ESI - MS/MS 



V° H. 



Ni-y^ 






C16H18N206S 

MW366.39 
[M+H]^ = 367.39 



\ 

C16H16N205S 

MW348.37 
[M+H]" = 349.38 




C10H7NO2 

MW173.17 

[M+Hr= 174.18 






C15H18N204S 

MW322.38 
[M+H]* = 323.38 



+ 



OH 

C6H11N04S 

MW193.22 

[M+H]*= 194.22 



H,C NH 

H3C ^ — OH 

O 

C6H9N02 

MW127.14 

[M+Hr= 128.15 



Figure B-22: The interpretation of the positive HPLC/ESI-MS/MS of compound 366a- 
penicillin G sulfone. 



.i * V 



•d 



■H 



165 



^m- 




el 

a 

w 

O 



•a 

ex 
o 

s 

o 

• ^^ 
I 

VO 
VO 



o 

I 

o 
o 

o 

1/3 



00 
00 

W 

U 

h-1 



douepunqv BAije|8y 



> 



o 

a, 

u 

H 

en 

I 

CQ 

i 



166 



366b - RT22.71 min / HPLC - ( + ) ESI - MS / MS 



HO 



-V^ 



o-f 




HjN* 



^ / 



C.sH.gNzOsS 

MW366.39 

\M+HY = 367.39 



i "''t 



H,C 



H,C 




H,N* 



\ / 



C,5H,gN204S 

MW322.38 

[M+H]" = 323.38 



HO 



H3C 



H3C 







o 

CsHgNO^S 
in/zl90.19 



CisH.eNzOsS 

MW348.37 

[M+H]" = 349.38 



+ 



HN* 




^ / 




C.oHtNOj 

MW173.17 

[M+Hr= 174.18 



HO, 



HoC 




NH* 



^t<r° 



C8H9O5S 

MW231.22 
[M+H]* = 232.23 



Figure B-24: The interpretation of the positive HPLC/ESI-MS/MS of compound 366b- 
isomer of penicillin G sulfone. 



167 




168 



366c - RT26.19 min / HPLC - ( + ) - ESI - MS / MS 




CsH.gNiOsS 

MW366.39 

[M+H]* = 367.39 




r\ 






C6H,N02S 

MW159.20 

[M+H]* = 160.21 



C.zHsNOjS 

MW231.27 

\M+HY = 232.28 






. CsHieNzOsS 
MW348.37 
[M+H]" = 349.38 



H3C^V5=N* 



CjHgNS 
m/zl 14.18 



N CH, 




CHj 




r\ 



hX 



C10H9NO4 

MW207.18 

[M+Hr = 208.19 



C,oH,N02 

MW175.19 

[M+H]*= 176.19 




'^Hj 



/ ^ 



CsHuNjOjS 

MW334.34 

[M+H]* = 335.35 



Figure B-26: The interpretation of the positive HPLC/ESI-MS/MS of compound 366c- 
isomer of peniciUin G sulfone. 



169 




souepunqv 9A|)e|9^ 







a3uepunqv 9A||e|3y 



o 

G 



•a 

a, 
o 



o 

M 
I 

(J 



o 

I 

o 
u 

o 

00 



czi 



00 

u 
h-1 



> 

el 

(U 

H 
CQ 

00 



170 



366c - RT26.25 min / HPLC - ( - ) ESI - MS / MS 



/ 




°. X 




HO 

CisHigNzOsS 

MW366.39 

[M-H]- = 365.38 



/ % 





CHa 
H3 



CsHmNzOjS 

MW292.39 

[M-H]- = 291.39 




CjHitNOzS 

MW251.34 

[M-H]- = 250.33 



H,c 




C6H9NO2S 

MWl 59.20 

[M-H]-= 158.19 



/ ^ 





CH, 
CH, 



0- 

CnHieNjOjS 

MW288.36 

[M-H]- = 287.36 



CH, 



//\ 




CH, 



Cl6H,6N204S 

MW332.37 
[M-H]- = 331.36 



Figure B-28: The interpretation of the negative HPLC/ESI-MS/MS of compound 366c- 
isomer of penicilhn G sulfone. 



.1 ■■ 



t ■ 






171 




souepunqv sAjieia^ 



. . 2ooo 
u. Et-oo 



Zco I— ULCO^tT 






t = cnoo 

■ 3 o T- oj 




o> o o o d> o 

O CO ID •* CM 

aouepunqv 9A!)E|9^ 



1/ 



•< 




E 
i- 



"^ 



3 



O 

•1—4 

o 



o 
00 



00 

I 

I— I 

00 
U 

H-1 



CO 

O 

o 

CQ 
o 

so 



172 



368 - RTl 1.64 min - HPLC / ( + ) ESI - MS / MS 




CeHsNOjS 

MWl 59.20 

[M+Hr= 160.21 




// 
o 

C,4H,3N203S 

ni/z289.33 



.s:!=-=CH, 




CisHjoNiOeS 

MW368.40 

[M+Hr = 369.41 




C,5H2oN204S 

MW324.39 
[M+H]^ = 325.40 




CsH.gNjOjS 

MW306.38 

[M+Hr= 307.39 



Figure B-30: The interpretation of the positive HPLC/ESI-MS/MS of benzylpenicilloic 
acid sulfoxide. 



''-yil-'i}r'.. 



APPENDIX C 

THE PRODUCTS OF RECOMBINATION REACTIONS OF PENICILLIN G 

FOLLOWING THE INTERACTION WITH POTASSIUM SUPEROXIDE - THE 

CHROMATOGRAMS AND MASS SPECTRA OF HPLC/ESI-MS/MS. 



o 
■0- 



CM 

o 



<s> 

a. 




SS8 

ffiSp 

*? CO 3O 



loKo 


Oc^O 


oinO 


J <mo 


S>"° 


-»So 


<D<isa 


1.41E6 
= 100.0-3 
-ull ms2 
00 - 420 


.32E 
100 

ull m 
0-4 


60E 
100. 
jll m 
0-4 


-- u U-H 


- Iiu-C! 


i^fi 


^f?2. 


^^."S 



^ 



.-" 



5< 




*?^ p> 



aouepunqv eAjieia^ 



o in 










JXITTTTT 

8 S 



Si 



l-H 



CM 


(U 




c 






CM 


g 




rt 










ot 





CM 0) 


C 


H 


<u 




ex 




IM 









C/1 


.<£> 






t/3 


,-* 


s 




l-H 




C/3 


-CM 


tiJ 




u 




hJ 




s 




c> 




> 








■u 








OT 


-<o 







cx 



lU 



U 



aouepunqv aAtiep^ 



b 



174 



175 



296 - RT4.39 min - HPLC / ( + ) ESI - MS / MS 



H3C- 



Hjn: 



CH3 s- 

/ 

-s 



OH 



-CH, 



NH, 



CH, 



OH 

C10H20N2O4S2 

MW296.40 

[M+Hr = 297.41 



H3C- 



CH3 s 




NH, 



OH 



CsHioNOiSj 
m/zl 80.26 



H3C 



=s=s 



C4S5NOS2 

MW147.21 
[M+H]^ = 148.22 




C,H,3N202S2 

ni/2245.33 



H3C 



H3C 



1 



C3H7S2 
m/zl 07.21 



H3C- 



H2N: 



CH3 s- 

/ 
-S 



OH 



-CH, 



CH, 



OH 



C,oH,7N04S2 

MW279.37 
[M+H]* = 280.37 



H3C- 



O^ 



H2N; 



CH3 s- 

/ 
-s 



OH 



-CH, 



CH, 



CoHisNOjSz 

MW261.35 

[M+H]^ = 262.36 



Figure C-2: The interpretation of the positive HPLC/ESI-MS/MS of penicillamine dimer. 



176 




o 



o 

!■ 

o 
o 

o 

GO 



I 
CO 

O 



_> 

O 

cx 

<L> 

H 

I 
U 
P 



b 



asuepunqv SAi|e|3y 



177 



440 - RT24.55 min - HPLC / ( + ) ESI - MS /MS 




C20H20N2O3 

MW336.39 

[M+Uy = 337.34 



OHO 




C22H20N2O6S 

MW440.47 
[M+H]+ = 441.48 



C2,H,6N205S 

MW408.43 
[M+H]*= 409.43 



o^> 

0: 





OH S^ 



OH 



C7H8N2O6S 

MW248.22 
[M+H]^ = 249.22 



HX 



H 



o h=^ 




// 

OH 

CgHioNCS 
in/z232.23 



■s^=CHj 



H3C 
H3C >=0 



■^ Si=CH2 

CgHioNOjS 
m/z200.23 



Figure C-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 440. 



178 



rf 




93uepunqv 3Ai)e|3^ 



,.. Booo 

LL tOOO 



jio O^ 



J5 

rs 




1 1 ' " I " " I ' 1 1 1 1 . 1 . . I 

3 Q O O C 

* <D lO ^ (» 

eauepunqv SAiieia^ 



m E 






o 

t 

o 
o 

o 

(/3 



CO 
I 

h- ( 

00 
U 

a 

(U 

> 



U3 

o 



I 

U 



b 



179 



632 - RT29.35 min - HPLC / ( + ) ESI - MS / MS 





MH HN — ( H3C- 

\ (^ \ — CH^Ha NH3 

0=/ SH3C 

OH 



C26H40N4O8S3 

MW632.80 
[M+H]*= 633.81 




CH, 



HjN 



-CH, 



S\. 



H3C- 



Q6H38N4O7S3 

MW6 14.79 

[M+H]* = 615.79 




CH, NH3* 



C10H20N2O4S2 

MW296.40 

[M+H]" = 297.41 





HJH*-( H,c- 
HjC— ^ y-CH^Hj NHj 
SHjC 

CnH33N305S3 

MW455.64 
[M+H]* = 456.65 




H^Hj nh; 



SHjC 



C,7H27N304S3 

MW433.60 
[M+H]" = 434.60 



Figure C-6: The interpretation of the positive HPLC/ESI-MS/MS of compound 632. 



180 



s 


A 


« 


SO 


■* in 


cn S 






^^1 


-"1 

Zri 6 




asuepunqv 3A||I:|3u 



o 

I 

o 
o 

o 

00 



00 



00 

U 

H-1 



> 

00 

ID 

H 
6 
bO 



181 



632 - RT29.20 min - HPLC / ( - ) ESI - MS / MS 




>-^ >-" 



O ) ( )— CHpH, NH, 

0=( SHjC 

O' 

C26H4oN408S3 

MW632.80 
[M-H]- = 631.79 



V— NH HN ( H»C-H (' 

o V— ( )~-<:hph, 

0=<^ SM,C 

O 

C26H3,N407S3 

MW614.79 
[M-H]- = 613.78 



m HN — ( H,c— ^ ' 

O \— ( )— CHpH, 



C25H40N4O7S3 

MW588.79 
[M-H]- = 587.78 




O \ ^ )— CHpH, 

0=^ SHjC 

C25H3,N405S3 

MW570.78 
[M-H]- = 569.77 




C,6H2oN204S 

MW336.40 
[M-H]- = 335.30 




C20H23N3O5S2 

MW451.53 

[M-H]- = 450.53 



9"= 



HN „ 



NH HN ( CHj 

0=< SHjC 



C22H3,N305S3 

MW517.71 
[M-H]-= 516.71 



+ 



CH, NH, 
C10H20N2O4S2 

MW296.40 
[M-H]- = 295.39 



^■. 



t 
CH. 

C6H,5NOS2 

MW181.31 
[M-H]- =180.31 



H,C CH, 




C2,H3,N304S3 

MW485.67 
[M-H]- = 484.66 



Figure C-8: The interpretation of the negative HPLC/ESI-MS/MS of compound 632. 



■ '4' 



182 



CM 
O 



o 

Q. 



Bo 
roZ CM £ 






s/ 



(0 



g» 



CD 

— I 9° 

g?o^ ■ 



gf 



o 



T"i 1 1 1 F I [ n 1 1 [ 1 1 
o o o 

CD lO TT 



i|i"i|iiiiim 



o 
-m 



oouepunqv aAfiejay 



UJ u_ Eooo 
Zoj h-Li.(£iT-r^ 



^' 




■■^■■■■^■■■■.^' 



aouepunqv 9Aj)e|9^ 



t I I I T r ITT p TT T T'TT t f J T I 1 I 




CO 



CO 



> 



o 

ex, 

H 

I 

U 

I 



183 



642 - RT30.13 min - HPLC / ( + ) ESI - MS / MS 




C3,H38N407S2 

MW642.78 
[M+H]^ = 643.79 




C.sHjoNjOjS 

MW308.39 

[M+H]^ = 309.40 




C22H33N3OSS2 

MW483.64 

[M+Hr= 484.65 




C22H3,N304S2 

MW465.62 
[M+H]* = 466.63 



Figure C-10: The interpretation of the positive HPLC/ESI-MS/MS of compound 642 



■■' > 



OW^A 



184 




aDuepunqv aAiie|9^ 



O 

a 

o 
o 

o 
on 



00 



en 
pq 

O 

H-1 



> 



U 



185 



642 - RT30.44 min - HPLC / ( - ) ESI - MS / MS 




C3,H38N407S2 

MW642.78 
[M-H]- = 641.78 




s — s 



V 



C30H38N4O5S2 

MW598.77 
[M-H]- = 597.76 




C31H36N4O6S2 

MW624.77 

[M-H]- = 623.76 



H,N 



% 





r^ 



C,8H,9N304S2 

MW405.48 
[M-H]- = 404.48 



Figure C-12: The interpretation of the negative HPLC/ESI-MS/MS of compound 642. 



APPENDIX D 

THE PRODUCTS OF CHEMILUMINESCENT REACTION OF 

PENICILLIN G FOLLOWING THE INTERACTION WITH POTASSIUM 

SUPEROXIDE - THE CHROMATOGRAMS AND MASS SPECTRA OF 

HPLC/ESI (OR APCI)-MS/MS. 



+ 

o 
a. 



o 
p 



^§ 



■<D 



OTT 

mo: 

Q.0O H" 

r? 

OS 



E 
t = 

ID =) 
C£) O 







^_ 


-o 

(D 






O)- 








lO 










O 






m 


-«) 






s- 


lO 






rj: 








in- 










o 






<D 








00 








■<t 








lO 








(D- 








•o- 






. ;-! ; '.• 


1- 


o 

-1? 






58^ 






"■ % ■ 


•<t 








•t. 
















■♦ 








(O 








t--- 








r) 


^ 






9 


CO 














o 








CTf= 








^ 








r 


^ 






00 


o 






O) 


-o 






^^-- 


CO 






CJ 








in - 








<£>._ 








00 CM 








lO 








'^S'l 


o 
-in 


- 




Cvl 


eg 






Br 


■ 








o 
-o 

CM 






IO_ 


. 






N- 








*■ _ 


- 






lO 










o 








-to 






p) 








5^_ 






f 


in 






^^ — 


o 






^" -3 


-o 




hJ 


*" 












11 \=.: 








CO " 




1 1 1 M 1 1 I M 1 1 1 M 1 MIT^riM 1 1 1 1 1 |l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 
3000000000< 


D 



eouepunqv gA|)e|9^ 




187 



188 



122 - RT1.71 min - HPLC / ( - ) APCI - MS 



// \J/ 



o 



O" 



C7H502 

MW122.12 
[M-H]-= 121.11 



Figure D-2: The interpretation of the negative HPLC/APCI-MS of benzoic acid. 



.r- a 



189 



C-\LCQ\dala\Sp«c\SEQ 1 979m 

nw chase (NH40AC -> MeOH) C18 HPLCA*)ESI-MS;MS 
S# MMa-i:ib ; R T : 33..tt>^.fe 7 AV: 10 NL 1.5<E6 
F- ♦ c Fu« ms2 337.00 ( 90.00 - 420.00) 



10/01/97 13:32:33 



P<3 ♦ K02. 1 mfl/mL 




'zio' ' '2^0' ' '260' ' '2to' ' '3A0' ' '3io' ' 'sio' ' '3^0' ' 'sio' 

m/z 



Rr:J2.G9. 36.12 
10CH 



7*: 



10^ 




Nl; 

2.92E« 

TlCFz + c 

FUiimZ 

337.00 ( 

90.00- 

420.001 



33^2 _ 34.24 3^.37 



328 330 33.2 3^.4 3^.6 ' 'lix ' 'M ' ii^' ' 'zi.* ' 3^.6' 

TliT»{mIn) 



_J476^^^ 35.11 35.,24 35.41 3i.56 35^1 M»1 



'3^.e' ' 3io' ' '3i2' ' '3i4' ' '3i.s ' 'six ' 'sio' 



C:\LCQ\dala\Spec\SEQ1979o 

rev phase (NH40Ac -> MeOH) C18 HPtC/(-)eSI-MS/MS 



10/01/97 15:47:44 



PG ♦ K02, 1 mfl/mL 



SH: 1137-1343 RT; 33 52-33 67 AV: 7 NL: 9J7E4 

F: - c Fun ms2 335 10 ( 90.00 - 42O.0O1 



80 

8 70-= 

c 

« 

■o 60- 

I 50 

0) 

i 40 
J2 
S. 30 

20- 



Mf^ 



158 



^ 



31S. 



335 



2io' ' '2io' ' 2^' ' '24o' ' '3io' ' 3^0' ' Mo' ' 'i^' ' 'sio' ' '460' ' '4io 
m/z 



100 120 140 160 1 



RT; 32.21 ■ 35,29 
lOH 

•0- 
70- 



32.<3 3256 .32,'3 32.M 33.10 3120 33. 



32 4 32 6 32 e 33 



f. 




NU 

2.60E5 

TlCF:-e 

FuiimZ 

U9.10I 

M.00- 

420.001 




2' ■ d.4' ■ '3i.6' ■ '3^.8 3^.0' U.i ' '3i.*~ Ut ' ' iit' '"'j^o' ' 'jij' 

TlnxtnWt) 



Figure D-3: The positive and negative HPLC/ESI-MS/MS of compound 336. 



.-iSf.; 



190 



336 - RT33.59 min - HPLC / ( + ) ESI - MS / MS 



H3C. 




CsHnNaOsS 

MW336.36 

[M+Hr= 337.37 





w 




s-^ 



nh; 



x;Hj 



C7H8N2O3S 

MW200.21 

[M+Hf = 201.22 



^\ 



H,C- 



\) 



HjN 



CHuNOjS 

MWl 89.23 

[M+Hr= 190.24 




H,C 



C,5H,4N204S 

MW3 18.34 
[M+Hr = 319.35 



HX 




CuHmNzOj 

MW2 18.25 

[M+Hr = 219.26 




C8H,N0 

MW135.16 

[M+Hr= 136.17 



Figure D-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 336. 



191 



336 - RT33.57 min - HPLC / ( - ) ESI - MS / MS 



H,C 
H,C 




.^-^ 



CftHsNOzS 

MW159.20 

[M-H]-= 158.19 




H3C 




-=0 




-^ 



C,5H,4N204S 

MW3 18.34 
[M-H]-= 317.34 



CuHuNjOj 

MW218.25 

[M-H]= 217.25 






NH .«- 



CsH.iNOjS 

MW177.22 

[M-H]-= 176.21 




\ 



H,C 




V 



C.sH.sNzOs: 

MW336.36 

[M-H]- = 335.35 



s 

// 




C7H8N2O3S 

MW200.21 

[M-H]- = 199.20 



158.19-.^^ 176.21 *- 




-H2O 



317.34 



199.20 - '"^^' '^"^ (231.24)^ 



Figure D-5: The interpretation of the negative HPLC/ESI-MS/MS of compound 336. 



'i yS>'' s t ' ■ 



192 








aouepunqv 9Ai)e|3^ 



T- (r 1 1 1 1 1 1 1 1 II 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 rin 

oiQOOOQQOOO 



93uepunqv sAiietdy 



193 



51 1 - RT31.07 min - HPLC / ( + ) ESI - MS / MS 




C2SH25N3O7S 

MW5 11.55 

[M+H]* = 512.55 



>* 'i: 




CoHtNOi 

MW173.17 
[M+H]*= 174.18 




C25H23N3O6S 

MW493.53 

[M+H]* = 494.54 



HaC^ 




CgHieNaOa 

MW308.33 

[M+H]* = 309.34 



Figure D-7: The interpretation of the positive HPLC/ESI-MS/MS of compound 511. 



APPENDIX E 

THE PRODUCTS OF UNKNOWN REACTIONS OF PENICILLIN G 

FOLLOWING THE INTERACTION WITH POTASSIUM SUPEROXIDE - THE 

CHROMATOGRAMS AND MASS SPECTRA OF HPLC/ESI-MS/MS. 



. ;. i 






o 

CO 



o 



E 

O) 

E 



O 



o 

Q. 



•i^ 



<6 



O 






05 



w o 
ii)0 

roZ 

Is 

0(0 




|iii'|iiii|ii"l""|iiii|ii M |iiii|iii i | n iii nn 
"ooooo--"^-- 
Oi CO t~ <o tn 



1 1 1 1 1 1 
o o o 

CO CM 1- 



(OU. 



aouepunqv 8/M)B|8a 



UJ U.<Mo 








^ 

;; 

a^ 










ON 



O 



^ fi 



o 
o 



00 

I 

I— I 

00 

u 
> 

60 

u 

(=1 
u 






aouepunqv SAi)e|3y 



At, 






196 



269 - RT22.69 min. - HPLC / ( - ) ESI - MS / MS 




S— CH, 

// ^ 



C,2H,5N04S 

MW269.31 
[M-H]- = 268.31 



/ \ 



u 
- 


^\ 


CnH 


UNO3S 


MW237.27 


[M-H]- 


= 236.26 



O N 




A 



S — CH, 



// 



C5H7NO4 

MW177.17 

[M-H]- =176.17 



N 



f 




=S=CH" 



CSH3NO2S 

MW141.14 

[M-H]-= 140.14 



Figure E-2: The interpretation of the negative HPLC/ESI-MS/MS of compound 269. 



•V ..; 



i > 



197 



o 



o 



CO- 



^ 



o 

-CM 
CO 



CO 



O 
O 



CO 



o 

I? 



a> 



So 

CO o 

oO 
a.-t 
Wi 



(0 



■o 
o 2 




r 
o 

. . + '" 
It .. 
(Ou. 



1 1] in I |i 1 1 1 1 1 1 1 1 |ni i| M III I III 1 1 1 

----- o o 



souepunqv sAiteis^ 



o 
-co N 

'1 



U-Oo 
OPCM 

UJ u."o 
ZcDH Era 



"-go 

S,i:"88' 

2(D I- EcoTf 




«i 



o E 

F 



s 



[,, o CO S -^ csj 
souepunqv SAqep^ 






O 

&, 

B 
o 
o 

o 

C/3 



00 

I 

I— ( 

w 

u 

h-1 



(U 

> 

• i-H 

o 

a, 
u 

H 

tn 
I 

W 

§ 

GO 



198 



353 - RT39.43 / HPLC - ( + ) ESI - MS /MS 




OH 

A 



-CH, 



CH, 



C,6H„N06S 

MW353.39 

[M+H]* = 354.39 




OH O 



CsHnNOsS 

MW335.37 

[M+H]* = 336.38 



OHjO^ 




OH 



CH, 



C7H10O5S 

MW206.21 
[M+H]* = 207.22 



\ 



-CH, 
CH, 

C7H10O3S 

MWl 74.21 
[M+Hr= 175.22 



' OH 



H2C, 



r 



-CH, 



CH, 



CeHiiOsS 
m/zl63.21 






CH, 
CH, 



C4H7OS 

m/zl03.16 



Figure E-4: The interpretation of the positive HPLC/ESI-MS/MS of compound 353. 



h J V 



,'.ff .■-"W^f"" ■■ 'VJ* ' 



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16. Halliwell, B. and Gutteridge, J. H. C. Biochem. J. 219: 1-14 (1984). 



l'l'^\>^ H^,\4. ■ ' ' '■'^■> ■' iw 



200 



17. Gartuer, A. and Weser, U. Top. Curr. Chem. 132: 1-61 (1986). 

18. Diguiseppi, J. and Fridovich, I. J. Biol. Chem. 257: 4046-4051 (1982). 

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20. Kallogg, E. W. and Fridovich, I. J. Biol. Chem. 252: 6721 - 6728 (1977). 

21 . Greenward, R. A. and Moy, W.W. Arthritis Rheum. 23 : 445 - 463 (1 980). 

22. Lunec, J., Halloran, S. P., White, A. G., Dormandy, T. L. J. Rheumatol. 8: 233-245 
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23. Snider. G. L. Am. Rer. Respir. Pis. 124: 321-324 (198n. 

24. Church, D. F. and Piyor, W.A. Environ. Health Perspect. 64: 1 1 1 - 126 (1985). 

25. Pryor, W. A. and Dooley, M. M. Am. Rev. Respir. Pis. 131: 941-943 (1985). 

26. Weisiger, R. A. Gastroenterology 90: 494-496 (1986). 

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28. Granger, P. N., Rutih, G., McCord, J. M. Gastroenterology 81: 22 - 29 (1981). 

29. McCord, J. M. and Roy, R. S. Can. J. Physiol. Pharmacol. 60: 1346-1352 (1982). 

30. Cerutti, P. A. Science 227: 375 -381 (1985). 

31. Zimmerman, R. and Cerutti, P. Proc. Natl. Acad. Sci. USA 81: 2085-2087 (1984). 

32. Weitzman, S. A. and Stossel, T. P. J. Immunol. 128: 2770-2087 (1982). 

33. Harman. P. J. Gerontol. 11: 298-300 (1956). 

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BIOGRAPHICAL SKETCH 

Jingshun Sun was bom in Jilin, China on October 7, 1967. After finishing his high 
school in 1986, he went on to undergraduate studies at Fuzhou University. He spent the 
next seven years of studying and researching in the field of Analytical Chemistry and 
received a Bachelor and Master of Science degree. Shortly after being employed in 
Fuzhou as a versatile employee learning about life, he was given the opportunity to join 
Dr. John H. Perrin's laboratory where he carried out the work described in this 
dissertation. In August of 1998, he will receive a Ph.D. in Medicinal Chemistry and move 
on to another challenging hurdle in life. 



207 



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I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




J6M H. Perrin, Chair 

Professor of Medicinal Chemistry 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Stephen U. Schulma 

Professor of Medicinal Chemistry 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




Lenneth B. Sloan 
Professor of Medicinal Chemistry 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 

Ian R. Tebbett 

Associate Professor of Medicinal 

Chemistry 



wV .y t t^J^* ! ' V «• .- ...• .1 i i 



I certify that I have read this study and that in my opinion it conforms to 
acceptable standards of scholarly presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 

h\4^ — <2— 




Jatfnes D. Wineforflner 
jraduate Research Professor of 
Chemistry 



This dissertation was submitted to the Graduate Faculty of the College of 
Pharmacy and to the Graduate School and was accepted as partial fulfillment of the 
requirements for the degree of Doctor of Philosophy. 



August 1998 

Dean, College' of Pharmacy 




Dean, Graduate School 






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UNIVERSITY OF FLORIDA 



3 1262 08555 3047